Novel process of solubilizing protein from a proteinaceous material and compositions thereof

ABSTRACT

The process for solubilizing proteinaceous material of the present invention includes subjecting the proteinaceous material to a sufficient amount of a basic solution to obtain a supernatant that has a basic pH and exposing the supernatant to the basic solution for a sufficient length of time and temperature for hydrolysis to occur. The process also includes cooling the mixture of the supernatant and proteinaceous material and optionally acidifying the mixture. This process may also include recovering the solubilized protein from the supernatant for use in various applications. Also provided herein is a composition of solubilized proteins from eggshell membrane obtained using processes of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. Ser. No. 12/253,719filed Oct. 17, 1998 which claims priority under 35 U.S.C. §119 of aprovisional application Ser. No. 60/980,607 filed Oct. 17, 1997, whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Many industrial waste products such as pigskin, fish scale, and avianeggshell membranes are a source of valuable bioactive materials,including collagen, that have widespread applications in medical, healthand cosmetic industries. To date, a major drawback to their use has beenthe difficulty in solubilizing these proteinaceous starting materials ina sufficiently stable and active pure form at an industrial scale sothat high yield is achieved in an economic manner.

For example, solubilization of eggshell membranes has proven technicallydifficult. Recent processes to solubilize eggshell membranes include theuse of mercaptopropionic acid, various extraction agents, or enzymes,such as peptidases, trypsin, and collagenases; however, problems havebeen associated with these procedures. The amount of protein solubilizedfrom the starting material by these processes is low, the techniques arenot cost-effective, and often the recovered protein components do notmaintain their native activity. Therefore an inexpensive process forsolubilizing eggshell membranes and other sources of proteinaceousmaterials while maintaining both yield, purity and activity of thesolubilized protein is needed, particularly one suited for commercialscale implementation.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a process to provide forcommercial-scale solubilization of various components from aproteinaceous material such as eggshell membranes. As a result of thepresent invention, one can produce a composition having large amounts ofsolubilized proteins from eggshell membranes. This process has anadditional advantage in that the resulting composition may be used as asource for the isolation of other valuable components. Specificcomponents, such as individual proteins, may be further purified fromthe composition thereby making it feasible to isolate proteins ofinterest from the composition.

The process for solubilizing proteinaceous material of the presentinvention includes subjecting the proteinaceous material to a sufficientamount of a basic solution for a sufficient length of time andtemperature for hydrolysis to occur. The process also includes coolingthe mixture of proteinaceous material/basic solution, and, if desiredacidifying the mixture, to obtain solubilized proteins. This process mayalso include recovering the solubilized proteins from the mixture foruse in various applications.

Therefore it is a primary object feature or advantage of the presentinvention to improve over the state of the art.

A further object, feature, or advantage of the invention is to provide anovel process for the solubilization of a proteinaceous material.

A further object, feature, or advantage of the invention is to provide aprocess for the solubilization of a proteinaceous material that producessolubilized protein that can be used in medical, cosmetic, pharmaceuticsdermatological or nutritional applications.

Another object, feature, or advantage of the invention is to provide aprocess for the solubilization of a proteinaceous material thatsubstantially lowers the mineral (ash) content of the solubilizedprotein composition.

Yet another object, feature, or advantage of the invention is to providea process for the solubilization of a proteinaceous material thatincreases the yield of the solubilized protein composition.

An object, feature, or advantage of the present invention is to providea means to solubilize proteins from eggshell membranes.

It is a further object, feature, or advantage of the present inventionis to provide a composition of solubilized proteins from eggshellmembranes.

Yet another object, feature, or advantage of the invention is to providea composition that is rich in proteins solubilized from eggshellmembranes.

Still another object, feature, or advantage of the invention is toprovide a cosmetic, medical, pharmaceutic, dermatological, ornutritional composition that is rich in proteins solubilized fromeggshell membranes.

An object, feature, or advantage of the present invention is to providea composition useful in treating an individual in need of proteinssolubilized from eggshell membranes.

An additional object, feature, or advantage of the present invention isto provide a method of treating an individual in need of proteinssolubilized from eggshell membranes.

A further object, feature, or advantage of the present invention is toprovide a process for preparing a composition that has solubilizedproteins obtained from an eggshell membrane.

A still further object, feature, or advantage of the present inventionis to provide a process which is suitable for implementation on acommercial/industrial scale.

One or more of these and/or other objects, features, or advantages ofthe present invention will become apparent from the specification andclaims that follow. No single embodiment of the invention need fulfillall or any of the objects stated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the bar graphs, the results for BIO1 are shown in the bar positionedto the left of a bar, which shows the results for BIO2.

As used herein, BIO1 refers to an eggshell membrane composition obtainedas a 3 kDa permeate using a 3 kDa membrane and nanofilter using themethods described herein. SKU313 and 314 refer to a spray dried form ofBIO1 and freeze dried form of BIO1. See also Example 5 for furtherdetails.

As used herein, BIO2 refers to an eggshell membrane composition obtainedas a 3 kDa retentate using a 3 kDa membrane using the methods describedherein. SKU309, OvaCore, BiOvaCore, OvaFlex, BiOva Flex, OvaDerm,BiOvaDerm all refer to a spray dried form of BIO2. See also Example 5for further details.

FIG. 1 is a flow chart for one embodiment of a process for solubilizingeggshell membranes.

FIG. 2 is a flow chart for one embodiment of a process for solubilizingeggshell membranes.

FIGS. 3A and 3B are flow charts for one embodiment of a process forsolubilizing eggshell membranes.

FIG. 4 is a graph showing the ability of the BIO1 and BIO2 fractions toinhibit the peroxyl free radical production by the enzymatic activity ofLipoxygenase.

FIG. 5 is a graph showing the ability of the BIO1 and BIO2 fractions toinhibit formation of Reactive Oxygen Species (ROS) in human PMN cellsunder normal culture conditions (A) and under conditions of oxidativestress (B).

FIGS. 6 A and B are graphs showing the ability of the BIO1 and BIO2fractions to support random migration behavior of human PMN cells. *BIO2was tested at one more serial dilution, due to extra plate space in allmigration experiments*

FIGS. 6 C and D are graphs showing the ability of the BIO1 and BIO2fractions to inhibit the directed migration of PMN cells towards theinflammatory chemoattractant Leukotriene B4 (LTB4). *BIO2 was tested atone more serial dilution, due to extra plate space in all migrationexperiments*

FIG. 6E is a graph showing the ability of the BIO1 and BIO2 fractions toinhibit the directed migration of PMN cells towards the inflammatorychemoattractant Interleukin-8 (IL-8). *BIO2 was tested at one moreserial dilution, due to extra plate space in all migration experiments*

FIG. 6 F is a graph showing the ability of the BIO1 and BIO2 fractionsto inhibit the directed migration of PMN cells towards the inflammatorychemoattractant f-MLP. *BIO2 was tested at one more serial dilution, dueto extra plate space in all migration experiments*

FIG. 7 are graphs showing the ability of the BIO1 and BIO2 fractions tosupport mitochondrial function under normal culture conditions (A) andunder conditions of oxidative stress (B). The testing was performedwhere each testing condition, including each serial dilution of testproduct, was performed in triplicate. The experiment was repeated threetimes using PBMC from different blood samples.

FIG. 8 are graphs showing the viability of cells under normal cultureconditions (A) in the presence of BIO1 and BIO2, and the ability of theBIO1 and BIO2 fractions to protect human PBMC from necrotic (C) andapoptotic (B) cell death under normal cell culture conditions.

FIG. 9 are graphs showing the viability of cells under oxidative stress(A) in the presence of BIO1 and BIO2, and the ability of the BIO1 andBIO2 fractions to protect human PBMC from necrotic (C) and apoptotic (B)cell death under oxidative stress.

FIG. 10A-F shows examples of data output from the Moritex/BTBP SkinAnalyzer. A dataset illustrated by the example above was collected ateach time point (clinic visit) and a progression of values was recorded.It was therefore possible to accurately quantify the changes in severalskin parameters measured in response to the daily application ofBiOvaDerm™ Cream.

FIG. 11 is a graph depicting the average percentage change from baselinewrinkle measurements for 8 subjects who used the cream for 4 weeks andwere followed an additional four weeks after stopping use.

FIG. 12 is a graph showing the Effect of 2 weeks Daily BiovaDerm Use onWrinkles and Redness.

FIG. 13 is a graph showing the average percentage change from baselinewrinkle measurements for 6 subjects who used the cream for 1 weeks andwere followed for another four weeks after stopping use.

FIG. 14 is a graph showing the Pore Health and Complexion relatedmeasurements over 4 weeks. [Read from “0” baseline to the vertical linewhich represents the stopping point]

FIG. 15 is a graph showing the Pore Health and Complexion relatedmeasurements over 2 weeks. [Read from “0” baseline to the vertical linewhich represents the stopping point]

FIG. 16 is a graph showing the Sum of wrinkle measurements(Deep+Fine+Emergent) for six subjects expressed as percentage of initialpre-use value. Cream use was started day 1 and discontinued after 2weeks Group 2. An overview of the effects of the treatment can be seenin the graph of Total Wrinkles index (Deep+Fine+Emergent) over thecourse of the study. The wrinkle data are plotted as percentages of thevalue calculated for the baseline scan taken during first visit 1 forthis group (i.e. before the subjects started the treatment). Subjectswere asked to discontinue use of creams at 2 weeks to test effect ofnon-use after daily application. The data for later visits representwrinkles analysis after product use had been discontinued. In thisgroup, use of the cream resulted in a reduction of wrinkles that wasdetected after 2 weeks of daily use and varied from 5% to 63% reductionamong the subjects. KEY: JR, LE, NR, RC=Initials of study subjects.

FIG. 17 is a graph showing the wrinkle measurements (Deep). Foursubjects expressed as percentage of initial pre-use value. Cream usestarted day 1 and discontinued at 2 weeks. In these subjects, use of thecream resulted in reduction of deep wrinkles detected after 2 weeks ofdaily use and varied 20% to 60% reduction among the subjects. When creamwas discontinued, the skin started to revert to its pre-treatment statewas seen over the following 4 weeks.

FIG. 18 is a graph showing the Wrinkle measurements (Fine) for foursubjects expressed as percentage of initial pre-use value.

FIG. 19: is a graph showing the Wrinkle measurements (Emerging) for foursubjects expressed as percentage of initial pre-use value.

FIG. 20 is a Joint Study Table which is further described in Example 14herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, processesand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.

To date, a composition containing proteins solubilized from eggshellmembrane has not been obtainable on a commercial scale due to lengthyprocedures, low yield of proteins and polysaccharides, or lack ofbioactivity of isolated proteins. There is no known process for thepreparation of a composition from eggshell membrane that overcomescurrent technical difficulties and produces a high yield, solubilizedprotein composition which is highly pure and undenaturated. Thesolubilized composition obtained by the process of the present inventionhas surprising levels of proteins, polysaccharides, and amino acids, isessentially free of odor, and can be efficiently prepared.Advantageously, since proteolytic enzymes or cross-linking agents arenot used, processes of the present invention are also more economic thanthose of current practices.

The process of the invention is suited for solubilization of proteinfrom various proteinaceous materials. Any suitable proteinaceousmaterial may be employed in the practice of the present invention. Asused herein, the term “proteinaceous material” is used to describe amaterial comprising proteins, polypeptides or peptides. Proteinaceousmaterials may be obtained and prepared from any number of resources.Examples include but are not limited to connective tissues, such as thecombs of roosters, avian eggshell membranes, skin, fishscale, flesh, orcartilage. As used herein, the term “eggshell membrane” refers to anypart of the eggshell membrane, for example, the inner eggshell membrane,the outer eggshell membrane or both. The eggshell membrane also includesthe eggshell membrane in its various forms, for example, frozen, raw(wet) or dried.

Avian eggshell membranes are conventionally considered a waste productdue in part to both the difficulties encountered in separating theeggshell membrane from the eggshell and further in the difficulties inprocessing the eggshell membrane in a manner that will result inobtaining proteins, polypeptides or peptides of interest. Variousmeasures of the protein content of eggshell membranes are present in theprior art.

Examples of one measure of the amount of protein and individual aminoacids known to be found in egg shell membranes are shown below from U.S.Pat. No. 6,899,294 to MacNeil.

Protein And Individual Amino Acids in Eggshell Membranes % Protein 85Lysine 3.35 Histidine 3.48 Arginine 6.46 Threonine 4.60 Glutamic Acid9.70 Proline 9.34 Glysine 4.94 Cysteine 8.50 Valine 6.30 Methionine 3.09Isoleucine 3.19 Leucine 4.30 Tyrosine 1.73 Phenylalanine 1.65

Another measure of the typical amino acid composition of egg shellmembranes is provided by U.S. patent application Ser. No. 10/797,747,published patent application no. 20040180025 (Long et al.) and is setforth in the below table:

Typical Amino Acid Composition of Eggshell Membrane Protein % Lysine2.88 Tryptophan 2.51 Leucine 3.85 Aspartic Acid 7.01 Proline 8.23Isoleucine 2.01 Threonine 4.42 Glycine 3.99 Histidine 2.79 Arginine 5.33Tyrosine 1.33 Glutamic Acid 8.23 Cystine 6.01 Alanine 2.00 Methionine2.85 Valine 5.13 Phenylalanine 1.48 Serine 4.28

Another measure of the constituents of egg shell membranes (inpercentage) is provided by Long et al and is set forth in the belowtable:

Typical Constituents of Eggshell Membrane % Collagen 35 Glucosamine 10Chondroitin 9 Hyaluronic acid 5-10

Prior testing on avian egg shell membranes with a particular process isdescribed in U.S. patent application Ser. No. 11/471,766, publishedpatent application no. 20070017447 entitled Avian Eggshell MembranePolypeptide Extraction Via Fermentation Process. That testing resultedin the following composition:

Constituent % Protein 90.08 Aspartic Acid 7.98 Threonine 5.19 Serine5.05 Glutamic Acid 11.91 Proline 10.79 Glycine 5.43 Alanine 2.46 Valine6.02 Isoleucine 2.91 Leucine 4.19 Tyrosine 1.57 Phenylalanine 1.60Lysine 3.21 Histidine 3.38 Arginine 6.89 Cystine 6.72 Methionine 3.50Tryptophan 3.64

Thus, there are numerous types of proteins, polypeptides, and peptidesin eggshell membranes that can be extracted provided that the membranescan be solubilized. The eggshell membrane may be obtained from anynumber of resources, including an egg-breaking facility. The eggshellmembrane may be separated from the egg white and eggshell using anysuitable technique, for example mechanical or chemical methods or acombination of methods. For example, unseparated eggshells may beprocessed as described in U.S. patent application Ser. No. 11/333,697,published application no. 20060159816, herein incorporated by referencein its entirety. The eggshell membrane separation method includesplacing the unseparated eggshells in a fluid tank containing a fluidmixture, such as a mixture of distilled water and acetic acid, andapplying cavitation to thereby assist in separating the eggshellmembranes from the eggshells. The eggshell membranes may then berecovered using any suitable technique, preferably the separationprocess does not damage or denature the proteins. The isolated eggshellmembrane may be processed as described herein. For example, the eggshellmembranes may be subjected to a solubilization process for solubilizingat least one type of polypeptide or polysaccharide from the eggshellmembranes. Components of interest that may be solubilized include butare not limited to collagen, elastin, desmosine, lysozyme, glucosamine,chondroitin, ovotransferrin, B-N-acetylglucosaminidase, hyaluronic acid,amino acids or other components of interest. The collagen may be Type Icollagen, Type V collagen, Type X collagen or combinations thereof. Thecomponents may be solubilized from the eggshell membrane and purifiedfor numerous uses.

As shown in FIG. 1, in one embodiment, the process of the presentinvention includes subjecting a proteinaceous material such as eggshellmembrane to a sufficient amount of a basic solution so that hydrolysisof the eggshell membrane occurs. The basic solution is added to theeggshell membrane to produce a supernatant having a basic pH. As usedherein, the term basic refers to a pH greater than 7. The pH of thesupernatant may be adjusted to a pH of from about 9.0 to a pH of fromabout 11.5, preferably from a pH range of from about 10.5 to about 11.5.Any suitable basic solution may be used including but not limited tosodium hydroxide, potassium hydroxide, and calcium hydroxide. Thesufficient amount of basic solution to add to the proteinaceous materialmay be determined in any number of ways as appreciated by those skilledin the art. For example, the sufficient amount of the basic solutionnecessary to achieve a pH from about 9.0 to about 11.5 may be determinedbased on the total weight of the proteinaceous material, preferablybased on the solid (dry) weight of the proteinaceous material, and themolarity of the basic solution. It is preferred that the temperature andpH be closely monitored, so that functional proteins, polypeptides andpeptides are obtained rather than mostly amino acids. As used herein, a“polypeptide”, “peptide” or “protein” are used interchangeably.

In one aspect, the process includes exposing the supernatant to thebasic solution for a sufficient length of time and temperature forhydrolysis to occur. One skilled in the art will appreciate that thetime needed for the hydrolysis reaction of the proteinaceous material toproceed will vary in part based on the temperature selected.Accordingly, hydrolysis of the proteinaceous material may be carried outat any suitable temperature for any suitable length of time, forexample, the temperature may be from a range of about 30° C. to about65° C., preferably from a temperature of about 45° C. to about 60° C.,and more preferably from a temperature of at least about 50° C. Thehydrolysis time can be as long as necessary to achieve the desiredresult. The length of time the proteinaceous material is subjected tohydrolysis may vary from as little as hours, such as 1 to 24 hours, todays depending on the temperature and other conditions used. Forexample, use of a higher temperature, for example of 50° C. compared to30° C., and stirring the mixture of proteinaceous material/basicsolution would reduce the length of time needed for hydrolysis reactionto occur. One skilled in the art can monitor the progress of thehydrolysis reaction using standard techniques such as trichloroaceticacid (TCA) protein precipitation and/or visualization methods. Forexample, one could take a sample of the supernatant, precipitateproteins out using TCA, and analyze the supernatant after TCAprecipitation for levels of nitrogen (indicative of free amino acids).Alternately, hydrolysis may be monitored by using simple visualizationmethods over selected time intervals. Typically, measurements areperformed about one to three hours after subjecting the proteinaceousmaterial to the basic solution to assess hydrolysis of the proteinaceousmaterial into soluble proteins. A sample may be taken from theproteinaceous material/basic solution mixture and the sample analyzedfor the presence of insoluble proteins of the eggshell membrane. Samplesare spun down using centrifugation and the contents visually analyzedfor the presence of a yellow material of non-hydrolyzed eggshellmembranes, i.e. insoluble eggshell membrane proteins. It is noted thatspun down samples may also contain eggshell particulates which are whitein color. If the yellow material of insoluble eggshell membrane proteinsis observed then the hydrolysis reaction is allowed to proceed andsamples from the mixture are taken at 15 to 20 minute intervalsthereafter and evaluated. Typically, the reaction is allowed to proceeduntil the yellow insoluble proteins are no longer present in the spundown samples. Thus, one skilled in the art can determine whether theproteinaceous material has been substantially hydrolyzed, whether moretime is needed or whether the reaction has proceeded too long asindicated by the hydrolysis of the proteins into amino acids.

In one aspect, the process of the invention includes cooling thesupernatant. For example, the temperature of the supernatant comprisingthe hydrolyzed proteinaceous material may be adjusted to a temperatureof from about 2° C. to about 18° C., more preferably to a temperature ofabout 2° C. to about 7° C.

In another aspect, the process includes the removal of particulates,such as eggshells or fine calcium from eggshells, from the supernatantcontaining the hydrolyzed proteinaceous material by any suitableseparation technique. This may be accomplished in any number of ways,including, but not limited to centrifugation, ultra-centrifugation,filtration or microfiltration, or combinations of separation techniques.For example, centrifugation may be used to separate particulates fromthe supernatant containing the hydrolyzed proteinaceous material and thesupernatant removed by decanting, pumping, and the like. Any number offiltration techniques may be used for the process of the presentinvention, including but not limited to gravity filtration, pressurefiltration, vacuum filtration, batch filtration, membrane filtration,filter press, continuous filtration, or any suitable combination.Filtration may include the use of any suitable filter that is capable ofremoving particulates from the supernatant. A suitable filter mayinclude but is not limited to a drum filter, a disk filter, filter pressor a sock filter. Preferably, a filter sock with a 100 micron to 865micron sock size is used. The filter may be made from a variety ofmaterials such as, but not limited to, sintered-metal, cloth, polymericfiber, natural fiber, paper such as a coffee filter, metal mesh, pulp,ceramic, or a combination of the foregoing materials, and the like. Thepore size of the filter may be of any size so long as it filters out thedesired particulates. The range of pore size may be from of 0.01micrometers to 100-200 micrometers, or greater.

The resulting solubilized components in the supernatant can be furtherpurified, isolated, and/or concentrated. For example, in one aspect, theprocess of the present invention includes removing salt (ash) orminerals from the supernatant. The relative amount of salts/minerals inthe supernatant can be determined using any suitable technique includingmeasuring the conductivity of the supernatant, using, for example, ameter to measure conductivity in milliSiemens (mS), ppm, ampre/volts,etc. The level of salt in the supernatant can be adjusted so that thefinal solubilized composition has the desired or acceptable level orpercentage of ash, depending on the intended use for the resultingsolubilized composition and the industry standards.

For example, with respect to the solubilization of avian eggshellmembranes, a conductivity of a supernatant that is at or above 5milliSiemens/cm may be considered to be a high level of salt/mineral. Ifthe salt/mineral content in the supernatant is not reduced, it willbecome ash in the resulting solubilized composition. Ash is undesirablebecause it is potentially perceived as a “filler” in the consumedproduct. Additionally, many health conscious consumers may desire tolimit their consumption of salt. The reduction in the amount of ash inthe supernatant increases the percentage of protein content in therecovered solubilized composition.

If the supernatant has a conductivity that would result in thesolubilized protein composition having an ash content that isunacceptable, salts/minerals may be removed from the supernatant untilan acceptable level of salt is present in the supernatant. In somecases, a supernatant having less than 5 milliSiemens/cm (mS/cm) isacceptable, preferably 4 or less mS/cm, more preferably from about 2 toabout 4 mS/cm.

The salt/minerals may be removed from the supernatant using any suitableprocess, for example, filtration, dialysis or ion exchange. The processmay include separating the hydrolyzed proteins in the supernatant fromsalt and if desired, specific molecules, using a membrane. Theseparation may be performed using any suitable technique, such as theuse of a membrane. This also allows for the concentration of acomposition that has high levels of solubilized proteins. Thecomposition may also contain polysaccharides. Any process that allowsfor concentration may be used, although, preferably the concentrationprocess maintains the biological activities of the composition or of theindividual components in the composition. Typically, the supernatantsare passed through a membrane having the desired nominal molecularweight cut-off value, leaving solubilized proteins and other solubilizedcomponents having a molecular weight larger than the cut-off valuebehind. In one embodiment, a membrane with a nominal molecular weightcut-off value of about 1000-3000 Daltons is used, resulting in acomposition that has high amounts of solubilized proteins, but allowingamino acids and other small molecules to pass through. For example, amembrane having a nominal molecular weight cut-off value of about 3kilodaltons may be used to isolate solubilized proteins larger than 3kilodaltons (BIO2, SKU309, Ovacore, Ovaflex, OvaDerm, 3 kDa retentate)and solubilized proteins that are less than 3 kilodaltons (BIO1, SKU313,SKU314, NF retentate) such as small molecular weight peptides in therange of 3,000 daltons to 400 daltons, which is the typical NF membranemolecular weight cutoff. If desired, the amino acids may be recoveredfrom the supernatant or retentate, such as a retentate produced by usinga nanofilter, for use in any number of applications, such as consumableproducts and compositions such as cosmetic, dermatological andpharmaceutical compositions.

Depending on the size of membrane used, the composition obtained fromthe retentate and/or permeate may also contain in addition to thesolubilized, hydrolyzed proteins and amino acids: acidglycosaminoglycans including but not limited to dermatan sulfate,chondroitin-4-sulfate (Picard, et. al., isolated and characterizedsulfated glycoproteins from eggshell membrane), glycoproteins includingbut not limited to hexosamines, hexoses, fucose; glucosamine, hyaluronicacid, ovotransferrin, desmosine, isodesmosine, lysyl oxidase, and/orlysozyme and the like.

Solubilized components may be isolated in any manner that is convenient.As appreciated by those ordinarily skilled in the art, the selection ofmembrane size can be used to obtain a composition enriched for aparticular size of protein or population of proteins. For example, useof a membrane having a nominal molecular weight cut-off value of about100 kDa may be used to isolate elastin and other solubilized proteinslarger than 100 kDa and proteins that are less than 100 kDa such ascollagen and desmosine. Desmosine, an anti-oxidant of four amino acidresidues of lysine, may be released when the elastin is solubilized, andif desired, may be further concentrated using a membrane having anominal molecular weight cut-off value of less than 500 molecular wt.Any suitable membrane size may be used such as 3, 6, 10, 50, 100, andgreater.

Advantageously, filtering and/or performing dialysis of the supernatantmay remove sulfur compounds from the supernatant, thereby reducing thesulfur odor of the supernatant. In another aspect, the process of theinvention may include removing odor causing components from thesupernatant, for example, by using a filter with an odor-absorbingcompound such as a charcoal filter or an activated carbon filter.Additionally, an odor-reacting compound that is an oxidizing agent, suchas hydrogen peroxide, may be added to the supernatant to reduce sulfurodors. The process may also include reducing the number ofmicroorganisms in the supernatant by subjecting the supernatant tofiltration, for example, a 0.8 micrometer filter.

In another aspect, the process includes adjusting the pH of thesupernatant or permeate comprising the hydrolyzed proteins so that thesupernatant or permeate has a pH from about 6.0 to about 8.0, preferablyto a pH of about 7.0. The pH may be adjusted using any suitable acidicsolution that has a pH of less than 7, including but not limited to asolution of acetic, oxalic, phosphoric, chloroacetic, citric, formic,benzoic, oxalic, succinic, acetic, propionic hydrochloric, nitric,sulfuric, hydrotropic, hydrologic, perchloric, chloric, phosphoric, orsulfurous acid or combinations thereof. In one embodiment, the pH andthe temperature of the supernatant or permeate are loweredsimultaneously or consecutively, although it is preferred that thesupernatant be cooled prior to addition of the acidic solution.

In a preferred embodiment, the removal of salts, for example, bydialysis, pH adjustment of the supernatant or permeate from a basic pHto a pH of about 7.0, and removal of sulfur odor using hydrogen peroxideare performed simultaneously. As appreciated by one skilled in the art,these steps may be performed consecutively, in a different order, oromitted and still yield a composition of solubilized proteins.

The solubilized composition resulting from the process of the presentinvention may be prepared in any number of forms or formulations. In oneembodiment, the composition of solubilized protein is prepared as aprotein powder using any suitable technique, including but not limitedto lyophilization, vacuum drying, freeze drying, spray drying, drumdrying, paddle-drying, super critical fluid processing, air drying, orother forms of evaporative drying. The drying step may be carried outany suitable temperature, for example, with respect to freeze drying, apreferred temperature range is from about 23° C. to about 40° C., with27° C. being the more preferred temperature.

The present invention is advantageous in that multiple components areefficiently and economically solubilized from the eggshell membrane atthe same time. Additionally, if desired, one or more specific componentsmay be isolated from the solubilized eggshell membrane, such as elastin,collagen or desmosine. Thus, the present invention allows for theproduction of a composition of a specific component or combination ofselected components in amounts suitable for use in a particularapplication. Thus, the composition may be customized for use in aparticular product, for example, a cosmetic product or a dietarysupplement. Advantageously, the compositions of the present inventionare essentially odor-free.

Once the proteinaceous material or source is solubilized, one skilled inthe art would be able to readily use standard biochemistry techniquessuch as membrane filtration or chromatography to isolate a protein ofinterest. Accordingly, the process of the invention may also includeisolating from the supernatant or dried solubilized composition variousproteins and polysaccharides of interest depending on the source of thestarting proteinaceous material. For example, proteins of interest thatmay be isolated from solubilized avian eggshell membrane include but arenot limited to elastin, desmosine, lysozyme, ovotransferrin,B-N-acetylglucosaminidase, collagen such as Type I collagen, Type Vcollagen, Type X collagen, or combinations thereof or other products ofinterest. Polysaccharides of interest that may be isolated include butare not limited to hyaluronic acid, glucosamine, and chondroitin.

In one embodiment, a soluble protein composition may be obtained frommethods of the present invention. In one aspect, the protein compositionmay include elastin, desmosine, ovotransferrin,B-N-acetylglucosaminidase, collagen such as Type I collagen, Type Vcollagen, and/or Type X collagen, amino acids or combinations thereof.The amino acids present in the composition may include tryptophan,cystine, methionine, aspartic acid, threonine, serine, glutamic acid,proline, glutamic acid, proline, glycine, alanine, valine, isoleucine,tyrosine, phenylalanine, lysine, histidine, arginine, hydroxyproline andthe like. The composition may also include hyaluronic acid, glucosamine,and chondroitin. In one aspect, the composition is more than 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% protein per weight ofcomposition. In one aspect, the composition includes at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%collagen and/or least 1% elastin. Of the collagen present, the collagenmay be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% of Type I collagen. The composition mayinclude 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% ofType V collagen. The collagen of the composition may be 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of Type X collagen. The amountof elastin in the composition may vary depending on the size of membraneused to isolate the solubilized proteins. As shown in FIGS. 2 and 3, thecomposition may contain at least 10%, 15%, 20%, 25%, 30%, 35% or even40% elastin. In one embodiment, the invention includes an isolated,soluble protein composition that is at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% soluble. The composition or protein is “soluble” or“solubilized” if at least at least 10% (by weight) of the protein orcomposition dissolves or does not aggregate in distilled water.Preferably, solubility of the protein or composition is assessed indistilled water, for example, at a concentration of 1 gram of theprotein or composition per 9 grams of distilled water. The compositionmay be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or 95% (by weight) water soluble. As discussed andshown elsewhere herein, the protein composition may also contain othereggshell membrane components including but not limited to acidglycosaminoglycans including but not limited to dermatan sulfate,chondroitin-4-sulfate, glycoproteins including but not limited tohexosamines, hexoses, fucose; glucosamine, hyaluronic acid,ovotransferrin, desmosine, isodesmosine, lysyl oxidase, and/or lysozymeand the like.

The failure to develop a commercially feasible process of solubilizingvarious components from eggshell membrane is due in part to failure byothers to demonstrate high yields of a product in a highly purified,soluble form and that has retained bioactivity. As used interchangeablyherein, a “bioactivity”, “biological activity” or “native activity”,refers to a function exerted by an intact, non-dentatured protein,polypeptide or peptide as determined in vivo, or in vitro, according tostandard techniques. The protein, polypeptide or peptide may behydrolyzed. As described herein, the compositions of the presentinvention may be obtained from eggshell membranes without the use ofproteolytic enzymes or cross-linking agents. Accordingly, thehydrolyzed, solubilized protein compositions are believed to besubstantially pure, undenatured and retain biological activity.

A method of the present invention includes treating an animal or humanin need of a component solubilized from eggshell membrane, e.g. protein,peptides, or amino acids, by administering a composition of the presentinvention. As described elsewhere herein, the composition, fractionthereof, or component thereof may exhibit one or more of the followingactivities: (1) antioxidant activity, (2) anti-inflammatory activity,(3) decreasing ROS formation, (4) maintaining mitochondrial functionunder conditions of ROS, (5) decreasing apoptosis under condition ofoxidative stress, (6) decreasing necrosis under condition of oxidativestress, (7) maintaining cell viability under condition of oxidativestress, (8) increasing PMN migration, (9) decreasing PMNB cell migrationtoward inflammatory mediator leukotriene B4, (10) anti-wrinklingactivity, (11) wound healing, (12) bone healing, (13) muscleregeneration, and (14) recovery after trauma. Accordingly, thecompositions, fractions or components, e.g. proteins or polysaccharidesisolated thereof, may be used in any number of applications, includingbut not limited to products or services in the cosmetic industry, see,for example, U.S. Pat. No. 7,169,379 to Kouzuki; products in the healthindustry, for example, products for use in joint health; and products inthe medical industry, such as for wound healing, see U.S. Pat. No.7,041,868 to Greene. Such applications are known in the art as well asthe appropriate techniques for inclusion in such applications.

Accordingly, the invention also provides a method of treating a varietyof diseases, disorders, and conditions that benefit from an effectiveamount of a composition of one or more components obtained fromsolubilized eggshell membrane, e.g. a hydrolyzed, water-soluble proteincomposition from the eggshell membrane, a composition of one of morefractions thereof such as a retentate fraction or permeate fraction ofcomponents having the desired molecular mass (kDa), or a compositioncomprising individual components, e.g. proteins or polysaccharides,thereof. As used here, the term “effective amount,” means that amount ofthe composition that provides a therapeutic benefit in the treatment,prevention, or management of one or more conditions, symptoms, diseases,and/or disorders. As used herein, unless otherwise defined inconjunction with specific diseases or disorders, the term “treating”refers to: (i) preventing a disease, disorder or condition fromoccurring in an animal or human that may be predisposed to the disease,disorder and/or condition but has not yet been diagnosed as having it;(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or (iii) relieving the disease, disorder or condition,i.e., causing regression of the disease, disorder and/or condition.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions ofthe disease, disorder or condition, diminishment of extent of disease,stabilized (i.e. not worsening) state of disease, disorder or condition,preventing spread of disease, delay or slowing of disease progression.“Treating” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment.

In general, the present invention provides compositions that havenumerous beneficial effects and a broad range of uses. For example,provided herein are methodologies for prophylactically treating, as wellas for therapeutically treating a variety of disesases, disorders, orconditions. In some embodiments, the disesases, disorders, or conditionsare associated with oxidative stress. As used herein,“oxidative-stress-associated disease, disorder, or condition” refers toa pathological condition of an individual that results at least in partfrom the production of or exposure to reactive oxygen species (ROS),including but not limited to apoptosis-induced oxidative stress,necrosis-induced oxidative stress, inflammation induced by oxidativestress, mitochondrial function modulated by oxidative stress. The term“oxidative-stress associated disease” encompasses pathological statesthat are recognized in the art as being conditions wherein damage fromfree radicals or reactive oxygen species is believed to contribute tothe pathology of the disease state, or wherein administration of a ROSinhibitor (e.g., antioxidant such as beta carotene), is shown to producea detectable benefit by decreasing symptoms, increasing survival, orproviding other detectable clinical benefits in treating or preventingthe pathological state.

Oxidative-stress-associated diseases, disorders, or conditions includebut are not limited to stroke, neurodegenerative diseases (such asAlzheimer's Disease, Parkinson's Disease, Huntington's Disease,spinocerebellar ataxias), trauma (such as spinal cord injuries, skeletalor cardiac muscle injuries, kidney injuries, or liver injuries),muscular disorders (such as mitochondrial myopathy, lactic acidosis),diabetes, ischemia-reperfusion tissue injury, hypoxic-induced tissuedamage, migraines, congenital mitochondrial diseases (such as MELAS,LHON, Kearns-Sayres Syndrome, MERRF, NARP, Leigh's Syndrome),neuromuscular degenerative disorders (such as Friedreich's Ataxia,Duchenne muscular dystrophy, Multiple Sclerosis), epilepsy, neuropathy,neurological and neuropsychological developmental delays, amyotrophiclateral sclerosis (Lou Gehrig's Disease), renal tubular acidosis, andaging related diseases or disorders (such as cognitive and motordisorders, progeria, cancer). For example, the compositions may beuseful in slowing down manifestations and symptoms of degenerativeaging, the pre-onset of Parkinson's disease, Alzheimer's disease,diabetes, and cancer. When the condition being treated in an individualis an inflammatory condition, the compositions may be useful in reducingany of the symptoms associated with inflammation, including, but notlimited to, inflammation, redness, pain, swelling, lameness, and loss ofmobility.

The invention also provides a method for treating the effects ofoxidative stress due to the production of harmful oxygen-derived specieswhich comprises administering an effective amount of a composition ofthe present invention to a mammal or human susceptible to oxidativestress. Such stress includes that due to oxidizing agents, increasedoxygen exposure, oxygen-induced degeneration or disease, reperfusioninjury, ionizing radiation, carcinogenic, chemotherapeutic, or mutagenicagents, aging, or arthritis and the like.

The present invention also provides methods for therapy and prophylaxisof oxidative-stress associated diseases, disorders or conditionscomprising administering to a patient a therapeutically-effective amountof a composition of the present invention. Exemplary disease orconditions states associated with oxidative-stress include withoutlimitation ischemic reperfusion injury, inflammatory diseases, systemiclupus erythematosus, myocardial infarction, stroke, traumatichemorrhage, brain and spinal cord trauma, Crohn's disease, autoimmunediseases (e.g., rheumatoid arthritis, diabetes), cataract formation,uveitis, emphysema, gastric ulcers, oxygen toxicity, neoplasia,undesired cell apoptosis, radiation sickness, and other pathologicalstates disclosed herein, such as toxemia and acute lung injury),cerebral ischemia, retinal ischemia, myocardial infarction, chronicheart failure, post-surgical cognitive dysfunctions, peripheralneuropathy, spinal cord injury, head injury, and surgical trauma. Suchdiseases, disorders or conditions can include “apoptosis-related ROS,”which refers to reactive oxygen species (e.g., O₂ ⁻, HOOH) which damagecritical cellular components (e.g., lipid peroxidation) in cellsstimulated to undergo apoptosis, such apoptosis-related ROS may beformed in a cell in response to an apoptotic stimulus and/or produced bynon-respiratory electron transport chains (i.e., other than ROS producedby oxidative phosphorylation). Methods of the invention include treatingcells that are apoptotic or otherwise undergoing programmed cell death,or at risk to undergo such programmed cell death by administering aneffective amount of a composition of the present invention such assolubilized eggshell membrane, fractions thereof or components, e.g.proteins or polysaccharides isolated thereof. Exemplary cells includebut are not limited to ventricular cells and neuronal cells. Forexample, an individual suffering from or susceptible to heart failurecan be treated in accordance with the invention. Methods for treating adisease, disorder, or condition resulting in part from apoptosis-inducedby oxidative stress such as from ROS are provided. Therefore, one aspectof the present invention relates to a method of decreasing oxidativestress-induced injury and/or death of a cell in an individual, forexample, as compared to a control not administered a composition of theinvention.

Additionally, a subject suffering from a condition involving programmedcell death of neuronal cells can be treated in accordance with theinvention, particularly to treat a subject suffering from stroke, spinalcord injury or a neurodegenerative disease such as Alzheimer's disease,Parkinson's disease, Huntington's disease, Amyotrophic LateralSclerosis, Down's Syndrome, Korsakoffs disease, cerebral palsy,age-dependent dementia, diabetes, including treatment of pancreatic betacells, apoptosis of which can be involved with a subject suffering fromdiabetes. Treatment methods of the invention include administration to amammal in need of such treatment a therapeutically effective amount ofone or more compositions of the invention. The invention also includestreatment of diabetes, including treatment of pancreatic beta cells. Theinvention also provides a method for inhibiting cell necrosis which canbe used to treat any condition wherein it is desirable to prevent celldeath through necrosis, especially under conditions of oxidative stress.

Accordingly, the present invention also provides a method of treating orpreventing a condition associated with oxidative stress-induced injuryand/or death. The methods described herein may include administering acomposition of the present invention or a pharmaceutical, cosmetic,medical or dermatological product comprising the composition. In somecases, the individual has a condition associated with oxidativestress-induced cellular injury and/or death or may be at risk fordeveloping an oxidative stress-induced cellular injury and/or death.Conditions to be treated or prevented in accordance with this aspect ofthe present invention are any condition, disease, disorder, ordysfunction that implicates ROS in the etiology of the condition,disease, disorder, or dysfunction. Exemplary conditions, diseases,disorders, and dysfunctions include, without limitation, an inflammatorycondition, an allergic condition or an auto-immune condition,inflammatory pulmonary disease or reactions (e.g., asthma, allergicrhinitis, chronic obstructive pulmonary disease, and adult respiratorydistress syndrome), inflammatory musculoskeletal disease or reaction(e.g., soft tissue rheumatism, exercise-induced injury, rheumatoidarthritis, psoriatic arthritis, osteoporosis and osteoarthritis),inflammatory gastrointestinal disease or urogenital reaction (e.g.,enterocolitis, gastritis, Crohn's disease, interstitial cystitis,vaginitis, and ulcerative colitis), autoimmune disease or reactions(e.g., inflammatory bowel disease, and psoriasis), transplantationrejection reactions, ischemia, cataract, corneal pathology, glaucoma,retinal degeneration, vitreal degeneration, atherogenesis, hypertension,diabetes mellitis, hypercholesterolemia, cigarette smoking, degenerativediseases of aging and cancer, immune deficiency, hyperimmunity,autoimmunity, neurodegeneration, aging, glomerular nephritis,respiratory distress syndrome, asthma, coronary thrombosis, burns,sunlight exposure, psoriasis, dermatosis, trauma, Parkinson's disease,neurotoxins, dementia, rheumatoid arthritis, diabetes, pancreatitis,endotoxemia, intestinal eschemia, cataracts, retinopathy, retinaldegeneration, arteriosclerosis, Fanconi's anemia, malaria, inflammation,ischaemia-reperfusion, drug toxicity, iron overload, nutritionaldeficiency, alcohol, radiation, cancer, aging, HCV infection and AIDS.Machoado-Joseph disease, multiple sclerosis, muscular dystrophy,senility, muscular atrophy, stroke, hepatopathies, systemic lupuserythematosus, mixed connective tissue disease, multiple sclerosis ordiabetes, stroke, neurodegenerative diseases (such as Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, spinocerebellarataxias), trauma (such as spinal cord injuries, skeletal or cardiacmuscle injuries, kidney injuries, or liver injuries), neuromusculardegenerative disorders (such as Friedreich's Ataxia, Duchenne musculardystrophy, Multiple Sclerosis), epilepsy, neuropathy, neurological andneuropsychological developmental delays, amyotrophic lateral sclerosis(Lou Gehrig's Disease), muscular disorders (such as mitochondrialmyopathy, lactic acidosis), diabetes, ischemia-reperfusion tissueinjury, hypoxic-induced tissue damage, migraines, congenitalmitochondrial diseases (such as MELAS, LHON, Kearns-Sayres Syndrome,MERRF, NARP, Leigh's Syndrome), renal tubular acidosis, and agingrelated diseases or disorders (such as cognitive and motor disorders,progeria, cancer). While the above list is merely illustrative, a morecomplete list of mitochondrial diseases or disorders that can be treatedin accordance with the present invention is provided in United StatesPatent Application 2001/0005719 by Von Borstel, which is herebyincorporated by reference in its entirety.

In some cases oxidative stress is due to the production of harmfuloxygen-derived species. Any disease, disorder, condition orphysiological and/or pathological situation leading to overproduction ofROS may be treated using the compositions and methods of the presentinvention. Numerous studies suggest that the aging process and variousdisease-related degenerative processes are caused, at least partly, bythe free-radical-mediated oxidative stress and/or the oxidative shift inthe thiol/disulfide redox state (Beckaman and Ames, Physiol. Rev. 78(1998) 547-581; Droge, Physiol. Rev. 2002, in press). Oxidative stresshas also been implicated in the development of neurodegenerativediseases, especially Alzheimer's disease (Montine et al., J.Neuropathol. Exp. Neurol. 56 (1997) 866-871; Sayre et al., J. Neurochem68 (1997) 2092-2097; Lovell et al., Neurobiol. Aging 18 (1997) 457-461;Multhaup et al., Biochem. Pharmacol. 54 (1997) 533-539; Pratico et al.,FASEB J. 12 (1998) 1777-1783; Behl et al., Cell 77 (1994) 817-827;Kaltschmidt et al., Proc. Natl. Acad. Sci. USA 94 (1997) 2642-2647), andamyotrophic lateral sklerosis (Rosen et al., Nature 362 (1993) 59-62; Tuet al., Lab. Invest. 76 (1997) 441-456). Moreover, studies on primatesrevealed a massive age-related increase in oxidative stress in theskeletal muscle tissue (Zainal et al.; FASEB J. 14 (2000) 1825-1836),arid clear manifestations of oxidative stress were also seen in geneexpression profiles of skeletal muscle tissue and brain tissue from oldmice as detected by oligonucleotide arrays (Lee et al., Science 285(1999) 1390-1393; Lee et al., Nature Genet. 25 (2000) 294-297. Exemplarydiseases and disorders that may be treated with a composition of theinvention include but are not limited to neurodegenerative diseases(such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,spinocerebellar ataxias) and/or dementia.

In some cases, the oxidative stress may in part result from normalmitochondrial function. For example, in mammals, the cellular levels ofreactive oxygen species (“ROS”) produced by normal mitochondrialfunction increase throughout the aging process (Passos et al,Mitochondria and ageing: winning and losing in the numbers game,BioEssays 29:908-917, 2007). In other cases, oxidative stress may resultfrom abnormal mitochondrial function causing disorders such asMitochondrial Encephalomyopathy Lactic Acidemia and Stroke-like episodesMELAS, Myoclonic Epilepsy with “Ragged Red” (muscle) Fibers) (MERRF),Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa (NARP),Leber's Hereditary Optic Neuropathy (LHON), Leigh's Syndrome (SubacuteNecrotizing Encephalomyopathy), Progressive External Opthalmoplegia(PEO), and Kearns-Sayres Syndrome, pigmentary retinopathy, ataxia, andheart-block. mitochondrial dysfunction is diabetes, type II diabetesmellitus, cardiomyopathy, Parkinson's disease, Huntington's disease, andpremature aging and the like. Accordingly, the invention also provides amethod for treating an individual having oxidative stress associatedwith normal or abnormal mitochondrial function by administering aneffective amount of a composition of the present invention. In apreferred embodiment, mitochondria-containing cells are contacted invitro or in vivo with a composition of the present invention. Exemplarycells include, without limitation, neuronal cells, muscle cells(preferably skeletal or cardiac muscle cells), liver cells, and/orkidney cells.

Oxidative stress and mitochondrial function can be determined in anynumber of ways, for example, measuring mitochondrial DNA damageincluding mitochondrial protein production, changes in mitochondrialoxidative phosphorylation or changes in mitochondrial ATP productionwould accomplish the same goal. See also, for example, U.S. Pat. No.7,288,374 to Pincemail, et al. and U.S. Pat. No. 7,267,946 to Runge etal. describing different methods for the detection of oxidative stressin an individual.

Inflammation may result in part from oxidative stress. Accordingly,further provided are methods for decreasing in an individual at risk foror having inflammation or an inflammatory response inflammation or aninflammatory response. The method includes administering to theindividual a composition comprising an effective amount of a compositionof the present invention sufficient to reduce inflammation or aninflammatory response. Accordingly, the invention provides compositionsand methods of inflammatory diseases or reactions. In some embodiments,these inflammatory diseases are disorders of the bone joint such asosteoarthritis, osteoporosis, rheumatoid arthritis, and soft tissuerheumatism. The methods of the present invention can also be used toprevent and/or treat inflammatory skin diseases (e.g., atopicdermatitis, eczema, contact dermatitis, allergic dermatitis), skinirritation, inflammatory pulmonary disease or reactions (e.g., asthma,allergic rhinitis, chronic obstructive pulmonary disease, and adultrespiratory distress syndrome), inflammatory musculoskeletal disease orreaction (e.g., soft tissue rheumatism, exercise-induced injury,rheumatoid arthritis, psoriatic arthritis, osteoporosis andosteoarthritis), inflammatory gastrointestinal disease or urogenitalreaction (e.g., enterocolitis, gastritis, Crohn's disease, interstitialcystitis, vaginitis, and ulcerative colitis), autoimmune disease orreactions (e.g., inflammatory bowel disease, and psoriasis), musclefatigue, osteoarthritis, rheumatoid arthritis, inflammatory bowelsyndrome or disorder, skin inflammation, such as atopic dermatitis,contact dermatitis, allergic dermatitis, xerosis, eczema, rosacea,seborrhea, psoriasis, atherosclerosis, thermal and radiation burns,acne, oily skin, wrinkles, excessive cellulite, excessive pore size,intrinsic skin aging, photo aging, photo damage, harmful UV damage,keratinization abnormalities, irritation including retinoid inducedirritation, hirsutism, alopecia, dyspigmentation, inflammation due towounds, scarring or stretch marks, loss of elasticity, skin atrophy,gingivitis, and transplantation rejection reactions. The inflammation orinflammatory response may be chronic or acute. The inflammatory responseis at least in part mediated by an antibody or at least in part mediatedby cellular immunity. The treatment results in a reduction in severityof a symptom of inflammation (e.g., swelling, pain, headache, fever,nausea, skeletal joint stiffness, or tissue or cell damage). In yetanother aspect, the treatment results in inhibition of antibodyproduction or lymphoid cell proliferation. The composition may beformulated in any suitable form that is suitable for delivery, e.g. apharmaceutically acceptable form with a carrier, excipient or adjuvant.In another embodiment, a composition is formulated for topicalapplication for local prevention of inflammation and/or tissue damageconsequent to inflammation (e.g., psoriasis, atopic dermatitis, etc.).As will be readily apparent to those of skill in the art, thecompositions of the present invention can be used on their own to treatinflammation or an inflammatory response or alternatively, they can beused in combination with other known anti-inflammatory agents.

A variety of steroidal and nonsteroidal anti-inflammatory agents can becombined with a composition of the present invention and used to treatinflammation. Examples of suitable steroidal anti-inflammatory agentsinclude, but are not limited to, corticosteroids, such ashydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionate, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortinebutylester, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fluradrenolone acetonide, medrysone,amcinafel, amcinafide, betamethasone and the balance of its esters,chloroprednisone, chloroprednisone acetate, clocortolone, clescinolone,dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,fluperolone, flupreclnisolone, hydrocortisone valerate, hydrocortisonecyclopentylpropionate, hydrocortamate, pammethasone, prednisolone,prednisone, triamcinolone, and mixtures thereof may be used.

Examples of suitable nonsteroidal anti-inflammatory agents include, butare not limited to, piroxicam, isoxicam, tenoxicam, sudoxicam,CP-14,304, aspirin, disalcid, benorylate, trilisate, safapryn, solprin,diflunisal, fendosal, diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acemetacin,fentiazac, zomepirac, clidanac, oxepinac, felbinac, mefenamic,meclofenamic, flufenamic, niflumic, tolfenamic acids, ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, tiaprofenic, phenylbutazone,oxyphenbutazone, feprazone, azapropazone, trimethazone and the like.Mixtures of these nonsteroidal anti-inflammatory agents can also beemployed, as well as the pharmaceutically-acceptable salts and esters ofthese agents. Of the nonsteroidal anti-inflammatory agents, ibuprofen,ketoprophen, naproxen, flufenamic acid, mefenamic acid, meclofenamicacid, piroxicam and felbinac are preferred and ibuprofen, naproxen, andflufenamic acid are most preferred.

In preferred embodiments, the methods and compositions of the inventionare used for treating (1) a skin disease, disorder, or condition such asaged skin related wrinkles, sun damaged skin, blocked pores, unevenpigmentation, skin roughness, acne, perfusion, and/or wounds, (2)musculoskeletal pain, musculoskeletal stiffness, decreased motion range,reduced mobility, (3) inflammation, (4) neurological damage such asParkinson's disease or Alzheimer's disease, (5) cardiac tissue necrosisresulting from cardiac ischemia, (6) autoimmune neurodegereration (e.g.,encephalomyelitis), (7) acute lung injury such as in sepsis andendotoxemia, (8) neuronal damage resulting from ischemia (e.g., stroke,drowning, brain surgery) or trauma (e.g., concussion or cord shock), (9)muscle tissue damage, muscle regeneration, muscle fatigue, (10),radiation-induced damage and/or (11) cystic fibrosis. For example, inone embodiment of the present invention, a method of treating anoxidative stress condition in an individual with cystic fibrosiscomprises the administering a composition an effective amount of acomposition of the present invention by any suitable delivery method orformulation.

In another embodiment, the invention includes a method of increasing ormaintaining the joint health of an individual, in some cases, underconditions of oxidative stress. Another aspect includes the treatment ofjoint diseases, disorders or conditions associated with inflammation,for example, synovitis, swelling, pain, headache, fever, nausea,skeletal joint stiffness, or tissue or cell damage, osteoarthritis,joint effusion, joint inflammation and pain, post operative arthroscopicsurgery, osteochondrosis dessicans (OCD), traumatic injury, fractures,degenerative joint disease (DJD), rheumatoid arthritis, psoriaticarthritis, synovitis, lameness, post operative arthroscopic surgery,deterioration of proper joint function, the inhibition of metabolicactivity of chondrocytes, and septic arthritis. Use of any of thecompositions of the present invention may diminish the inflammatoryresponse in the affected area so that joint health of an individual isrestored or the affects of inflammation alleviated. In one embodiment,about 150 mg of BIO2 is administered three times a day for effectivetreatment of musculoskeletal pain, musculoskeletal stiffness, decreasedmotion range, reduced mobility.

Other dosages of composition administered is from about 100 mg/day toabout 1000 mg/day. Thus it is contemplated that one may use, ranges fromabout 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950mg/day to about 1000 mg/day. The dosage may be taken once a day or 2, 3,4, or more as appropriate to treat the disease, disorder or condition.It will be understood that the exact method of administration anddosages of administration will be decided and adjusted at the time ofadministration, depending on the individual needs of a subject, takinginto consideration factors such as, age, disease, gender, performancestatus, etc., and such adjustments will be made by a trained physician.Therefore, the invention is in no way limited by the doses set forth.

According to another aspect of the invention, compositions of theinvention may be used for general care, as well as treatment andprevention of diseases, disorders, and conditions of the skin. Thecompositions according to the present invention may be used fortreatment of any disease, disorder or condition that affects the skinfor example, wrinkles, including both fine superficial wrinkles andcoarse deep wrinkles, skin lines, facial frown lines, expression lines,rhytides, dermatoheliosis, photodamage, premature skin aging, crevices,bumps, pits, large pores (e.g., associated with adnexal structures suchas sweat gland ducts, sebaceous glands, or hair follicles),“orange-peel” skin appearance, dryness, scaliness, flakiness and/orother forms of skin unevenness or roughness; blemishes such as acne,pimples, breakouts; excess skin oil problems such as over production ofsebum, oiliness, facial shine, foundation breakthrough; abnormaldesquamation (or exfoliation) or abnormal epidermal differentiation(e.g., abnormal skin turnover) such as scaliness, flakiness, keratoses,hyperkeratinization; inadequate skin moisturization (or hydration) suchas caused by skin barrier damage, environmental dryness; loss of skinelasticity (loss and/or inactivation of functional skin elastin) such aselastosis, sagging (including puffiness in the eye area and jowls), lossof skin firmness, loss of skin tightness, loss of skin recoil fromdeformation; non-melanin skin discoloration such as undereye circles,blotching (e.g., uneven red coloration due to, e.g., rosacea),sallowness (pale color), discoloration caused by telangiectasia orspider vessels; melanin-related hyperpigmented (or unevenly pigmented)skin regions such as age spots (liver spots, brown spots) and freckles;post-inflammatory hyperpigmentation such as that which occurs followingan inflammatory event (e.g., as an acne lesion, in-grown hair,insect/spider bite or sting, scratch, cut, wound, abrasion, and thelike); atrophy such as, but not limited to, that associated with agingor steroid use; other histological or microscopic alterations in skincomponents such as ground substance (e.g., hyaluronic acid,glycosaminoglycans, etc.), collagen breakdown and structural alterationsor abnormalities (e.g., changes in the stratum corneum, dermis,epidermis, the skin vascular system such as telangiectasia or spidervessels); tissue responses to insult such as itch or pruritus; andalterations to underlying tissues (e.g., subcutaneous fat, cellulite,muscles, trabeculae, septae, and the like), especially those proximateto the skin, smoothing uneven or rough skin, soothing itchy, inflamed orirritated skin, improving skin atrophy, skin blemishes, acne erythema,acne, photodamage, atopic dermatitis, restoring or rejuvenating agedskin, damaged, scarred skin, lightening hyper-pigmented skin, increasingthe rate of healing of wounds in or on the skin, as well as skin changedor damaged by extrinsic factors such as sunlight, radiations, airpollution, wind, cold, dampness, heat, chemicals, smoke, cigarettesmoking. The compositions may be used for the treatment and preventionof various cosmetic conditions and dermatological disorders, forexample, inflammation of the skin such as atopic dermatitis, contactdermatitis, allergic dermatitis, skin irritation, wrinkles,thin-skinning and the like. The term “wound” includes skin cuts, tears,lacerations, ulcers, abrasions, burns, punctures and the like in or onthe skin. As shown in Example 11, the composition of BiovaDerm™ (BIO2)provide numerous benefits in the treatment of various cosmeticconditions and dermatological disorders such as wrinkles, sun damagedskin, blocked pores, uneven pigmentation, skin roughness, acne, andperfusion.

The compositions of the invention may also be applied to the skin, hairor nails for the treatment of changes associated with aging of skin,nail and hair; uneven and rough surface of skin; acne; irritation;dermatoses; eczema; psoriasis; itchy scalp and skin; warts; herpes; agespots; lentigines; melasmas; blemished skin; mottled skin;hyperkeratosis; hyperpigmented skin; skin thickening due to elastosis ofphotoaging; abnormal or diminished syntheses of collagen,glycosaminoglycans, proteoglycans and elastin as well as diminishedlevels of such components in the dermis; stretch marks; skin lines; finelines; wrinkles; thinning of nail plate and hair; loss or reduction ofnail and hair resiliency, elasticity and recoilability; lack of skin,nail and hair lubricants and luster; dull and older-looking nail andhair; fragility and splitting of nail and hair; and the like.

Thus in one aspect, the invention provides a method for regulating askin condition by administering to the individual in need of treatmentthereof a composition of the present invention in an effect amount totreat the condition. Regulating a skin condition includesprophylactically regulating and/or therapeutically regulating skincondition, including visible and/or tactile discontinuities in skin suchas, but not limited to, regulating visible and/or tactilediscontinuities in the texture of skin, reducing post-inflammatoryhyperpigmentation, regulating non-melanin discoloration of skin,regulating moisturization and barrier properties of skin, regulatingepidermal differentiation of skin, regulating exfoliation of skin,thickening of skin to reduce skin atrophy, regulating the elasticity ofskin, reducing oily skin, regulating cellulite in skin, regulatingpruritus in skin, and promoting wound healing in skin and decreasingscarring. As used herein, prophylactically regulating skin conditionincludes delaying, minimizing and/or preventing visible and/or tactilediscontinuities in skin. As used herein, therapeutically regulating skincondition includes ameliorating, e.g., diminishing, minimizing and/oreffacing, discontinuities in skin. “Regulating the signs of skin aging”includes prophylactically regulating and/or therapeutically regulatingone or more of such signs (similarly, regulating a given sign of skinaging, e.g., lines, wrinkles or pores, includes prophylacticallyregulating and/or therapeutically regulating that sign). As used herein,prophylactically regulating such signs includes delaying, minimizingand/or preventing signs of skin aging. As used herein, therapeuticallyregulating such signs includes ameliorating, e.g., diminishing,minimizing and/or effacing signs of skin aging.

“Signs of skin aging” include, but are not limited to, all outwardvisibly and tactilely perceptible manifestations as well as any othermacro or micro effects due to skin aging. Such signs may be induced orcaused by intrinsic factors or extrinsic factors, e.g., chronologicalaging and/or environmental damage (e.g., sunlight, UV, smoke, ozone,pollutants, stress, etc.). These signs may result from processes whichinclude, but are not limited to, the development of texturaldiscontinuities.

The compositions used in the methods of the invention may be “personalcare products” such as health and cosmetic beauty aid products generallyrecognized as being formulated for beautifying and grooming the skinand/or hair. For example, personal care products include anti-wrinklecream or acne products (e.g., lotions, skin creams, etc.), cosmetics,toiletries, and over-the-counter pharmaceutical products intended fortopical or oral usage.

As described elsewhere, the composition of the present invention can beformulated for topical application. In a preferred embodiment, acomposition of the present invention is added to a topical cosmeticformulation to diminish wrinkles and/or treat acne. In one embodiment,one FTU of about 10% weight cream of BIO2 is administered topically,preferably twice a day to treat wrinkles, aged skin, blocked pores,uneven pigmentation, skin roughness, acne, and/or perfusion. Otherdosages of composition administered is from about 5% weight to about 10%or 15% weight of the composition of the invention in the carrier. Thusit is contemplated that one may use, ranges from about 5% weight, about10% weight, about 15% weight, 20% weight, about 25% weight, about 30%weight, 35% weight, 40% weight, about 45% weight, about 50% weight, toabout 60% weight of the composition of the invention to the carrier suchas a cream. The dosage may be taken or applied once a day or 2, 3, 4, ormore as effective to treat the disease, disorder or condition. It willbe understood that the exact method of administration and dosages ofadministration will be decided and adjusted at the time ofadministration, depending on the individual needs of a subject, takinginto consideration factors such as, age, disease, gender, performancestatus, etc., and such adjustments can made by one skilled in the art.Therefore, the invention is in no way limited by the doses set forth.

Agents can be added to the topical formulation for such secondarypurposes as treating skin dryness, providing protection against light;other medications for treating dermatoses, preventing infection,reducing irritation, inflammation and the like. For example, products ofthe invention may include, in addition to a composition of theinvention, other dermatologically active compounds and materials,including without limitation, sunscreen ingredients, alpha hydroxyacids, retinoids, beta hydroxy acids, anti-inflammatories,antibacterials, antifungals, antioxidants hydroxyacids, ketoacids andrelated compounds; phienyl alpha acyloxyalkanoic acids and derivativesthereof N-acetyl-aldosamines, N-acetylamino acids and related N-acetylcompounds; those that improve or eradicate age spots, keratoses andwrinkles; local analgesics and anesthetics; antiacne agents;antibacterials; antiyeast agents; antifungal agents; antiviral agents;antidandruff agents; antidermatitis agents; antihistamine agents;antipruritic agents; antiemetics; antimotionsickness agents;anti-inflammatory agents; antihyperkeratolytic agents; antiperspirants;antipsoriatic agents; antiseborrheic agents; astringents; cleansingagents; hair conditioners and hair treatment agents; antiaging andantiwrinkle agents; sunblock and sunscreen agents; skin lighteningagents; depigmenting agents; vitamins; corticosteroids; tanning agents;hormones; retinoids; topical cardiovascular agents; corn, callus andwart removing agents; and other.

When the compositions according to the present invention are used fortreating skin wounds, for example in aiding the healing of skin cuts,tears, lacerations, burns, punctures, and other wounds, examples ofsuitable cosmetic or other agents that may be combined with one or moreoligosaccharide aldonic acids or related compounds include:hydroxyacids, ketoacids and related compounds; phenyl alphaacyloxyalkanoic acids and derivatives thereof N-acetyl-aldosamines,N-acetylamino acids and related N-acetyl compounds; analgesics andanesthetics; wound cleansers; antibacterials; antiyeast agents;antifungal agents; antiviral agents; anti-inflammatory agents; skinlightening agents; depigmenting agents; vitamins; burn relief agents;and corticosteroids. The administration of one or more compositions oragents, in accordance with the methods of the invention may occurtogether, concurrently, separately, sequentially, or a combinationthereof. One skilled in the art is well versed in the art of testing forwound healing. See, Shimamura K, Nakatani T. Ueda A, Sugama J. Okuwa M.Relationship between lymphangiogenesis and exudates during thewound-healing process of mouse skin full-thickness wound. Wound RepairRegen. 2009 July-August; 17(4):598-605.

The compositions of this invention may be delivered topically by anymeans known to those of skill in the art that is suitable for deliveryof the composition. The composition of this invention may furtherinclude a pharmaceutically, dermatologically, or cosmetically acceptablevehicle capable of functioning as a delivery system to enable thepenetration of the composition into the skin or wound. Thus, anycomposition of the invention may be formulated in the form of asolution, suspension cream, oil, gel, liquid, emulsion, ointment, salve,a powder, a spray or other forms known to those of ordinary skill in theart such as a wound dressing, an adhesive-containing bandage, a dermalpatch, a cotton roll bandage and the like. Preparation of such topicalformulations are well described in the art of pharmaceuticalformulations as exemplified, for example, by Remington's PharmaceuticalScience, Edition 17, Mack Publishing Company, Easton, Pa.

The compositions of this invention may also be administered by otherroutes as described elsewhere herein, for example, orally. Preferably,the compositions of the invention are applied to the skin over a periodof time sufficient to affect skin-related processes, e.g., reducewrinkles or repair sun-damaged skin. The methods of application in thepresent invention will depend on the ultimate intended use of thecompositions. Generally, the effects of the application and use of thecompositions of this invention become visible after approximately 4weeks of treatment. It is preferable to apply the compositions of thisinvention in an effective amount once or twice per day. In one specificembodiment of the present invention, the topical cosmetic composition ofthe present invention can be formulated as a night cream or a nightrepair serum, which can be applied to the face of an individual beforesleep or a period of bodily rest. In another specific embodiment of thepresent invention, the composition of the present invention isformulated as a facial mask, which can be applied to the face beforesleep or bodily rest, left thereon for a sufficiently long period oftime (e.g., overnight), and then rinsed off. The topical cosmeticcompositions can be applied locally to selected skin areas in need oftreatment. The topical cosmetic compositions can also be appliedgenerally to facial skin or other parts of the human body to achieve thedesired effects in such parts. The topical cosmetic compositions of thepresent invention may be applied to the skin on an as-needed basis, oraccording to a pre-set schedule. The topical cosmetic compositions ofthe present invention may be applied directly to clean skin, beforeapplication of any moisturizer, foundation, make-up, etc. Alternatively,such compositions can be applied over moisturizer, and optionally overfoundation and/or make-up. The amount applied each time, the area ofapplication, the duration of application, and the frequency ofapplication can vary widely, depending on the specific need of the user.For example, the topical cosmetic compositions can be applied for aperiod of days to months or even years, and at a frequency ranging fromabout once per week to about five times per day. For another example,the compositions can be applied for a period of about six months and ata frequency ranging from about three times a week to about three timesper day, and preferably about once or twice per day.

In some cases, such as strenuous exercise or injury, the level ofreactive oxygen species (ROS) produced in muscle tissue may be elevatedto a level that causes damage to muscle tissue such as skeletal muscle.Provided by the invention are methods for reducing or inhibiting muscletissue damage resulting from oxidative stress, for example, from ROS.The method includes administering to a subject/individual a compositioncomprising an effective amount of at least one composition of thepresent invention. Further provided are methods of preservingmitochondrial function in muscle tissue exposed to oxidative stresscomprising the administration to a subject a composition comprising aneffective amount of at least one composition of the present invention.In one example, an effective amount can inhibit or decrease theformation of reactive oxygen species and slow the progression ofoxidative stress in muscle tissue that are exerted or are afflicted by adisorder. In another aspect, an effective amount can reduce, decrease,inhibit or abrogate muscle tissue damage due to reactive oxygen species(ROS), preserve mitochondrial function in muscle tissue exposed to ROS;and/or improve, prevent or rectify motor performance and/or muscleendurance in physiological conditions where such functions arecompensated or a combination of these activities. Detection of ROS andRNS production muscle cells can be performed by various techniquesincluding electron spin resonance, fluorescent assays, cytochrome creduction, chemiluminescence, hydroxylation of salicylate, and assays tomeasure nitration of phenylalanine or arginine.

The invention includes treating diseases, disorders, or conditions suchas muscle injuries, muscular dystrophies, neuromuscular diseases,myasthenia gravis, multiple sclerosis, amyotrophic lateral sclerosis,age-related sarcopenia, muscle fatigue, decreased muscle endurance, lossof muscle function and the like in an individual in need of suchtreatment by administering an effective amount of a composition of thepresent invention. The methods may also be used to restore musclehealth, for example, from muscle damage from injury or exercise, or toretain muscle health. In another aspect, the methods may be used forenhancing motor performance and/or muscle endurance in an individualcomprising administering to a subject an effective amount of at leastone composition of the present invention. Examples of such conditioninclude muscular dystrophies, muscle injuries, neuromuscular disorderssuch as myasthenia gravis, multiple sclerosis, amyotrophic lateralsclerosis, age-related sarcopenia, muscle tissue wasting, decreasedenergy and immune impairment and the like. One skilled in the art wouldbe familiar with techniques for evaluating the compositionseffectiveness in treating these diseases, disorders, or conditions. See,for example, Moresi V, Garcia-Alvarez G, Pristera A, Rizzuto E,Albertini M C, Rocchi M, Marazzi G, Sassoon D, Adamo S, Coletti D.Modulation of caspase activity regulates skeletal muscle regenerationand function in response to vasopressin and tumor necrosis factor. PLoSOne. 2009; 4(5):e5570. Epub 2009 May 18.

Several routes for administration of the composition are contemplatedand these include but are not limited to oral, intramuscular,parenteral, or intrathecal. Other suitable routes are describedelsewhere herein. For example, the compositions may be consumed as afood, beverage, supplement, including sports food and/or drinks and feedfor animals including pet food. Exemplary forms include but are notlimited to bars, tablets, gels. pills or capsules. In addition tonutritional products suitable for consumption of humans, it is alsopossible to use at least one composition in feed for animals, such aslivestock or companion animals.

In some embodiments, the dosage of composition administered is fromabout 100 mg/day to about 1000 mg/day. Thus it is contemplated that onemay use, ranges from about 100 mg/day, 150 mg/day, 200 mg/day, 250mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850mg/day, 900 mg/day, 950 mg/day to about 1000 mg/day. Intermediate rangesare also contemplated, for example one may use 110 mg/day, or 270mg/day, or 365 mg/day or 576 mg/day and so on. It will be understoodthat the exact method of administration and dosages of administrationwill be decided and adjusted at the time of administration, depending onthe individual needs of a subject, taking into consideration factorssuch as, age, disease, gender, performance status, etc., and suchadjustments will be made by a trained physician. Therefore, theinvention is in no way limited by the doses set forth.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is diagnostically ortherapeutically effective. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. Local or regional administration, withrespect to an inflamed muscle, also is contemplated. Finally, systemicadministration may be performed. Delivery via syringe or catherizationis also contemplated.

Embodiments of the invention also relate to methods for treating and/orpreventing tissue and cell damage caused by reactive oxygen species(ROS) in mammals. More specifically, embodiments of the invention relateto the prevention and/or reduction of bone tissue and bone cell damagethrough the administration of a composition of the present invention.

In some embodiments, the invention described herein relates to methodsfor treating and/or preventing bone tissue and cell damage caused byreactive oxygen species in mammals, including humans. More specifically,the disclosure relates to the prevention of bone cell death and boneresorption through the administration of a composition of the presentinvention. In other embodiments, the invention relates to methods forreducing or preventing cell death or apoptosis in bone cells.

Bone cells include, for example, osteoclasts, osteoblasts, andosteocytes. In one embodiment, a method for treating or preventingreactive oxygen species (ROS)-mediated oxidative damage to bone cellsand tissues of a subject is provided, comprising the step ofadministering a composition of the present invention that reduces theamount of ROS to a subject suffering from or at risk for a bone diseasecaused or exacerbated by ROS-mediated oxidative damage. In someembodiments the ROS-mediated damage is enzymatically produced damage. Inalternative embodiments, the ROS-mediated damage is cellular derived,such as osteoclast-derived damage. In some embodiments, the amount ofROS is reduced by inhibiting the production or release of ROS. Althoughthe compositions and methods are applicable to any bone disease, theyare particularly relevant to the treatment of bone diseases selectedfrom the group consisting of osteoporosis, including, but not limitedto, type I and type II osteoporosis, age-related osteoporosis, disuseosteoporosis, diabetes-related osteoporosis, and steroid-relatedosteoporosis, periodontal disease, osteopenia, osteomalacia, osteolyticbone disease, primary and secondary hyperparathyroidism, multiplemyeloma, metastatic cancers of the bone, for example, of the spine,pelvis, limbs, hip, and skull, osteomyelitis, osteoclerotic lesions,osteoblastic lesions, fractures, osteoarthritis, infective arthritis,ankylosing spondylitis, gout, fibrous dysplasia, and Paget's disease ofthe bone.

Subjects suffering from or at risk for bone loss can be identified bymethods known in the art, such as, for example, by radiographicmeasurement of bone density, by evaluation of biochemical markers suchas alkaline phosphatase, osteocalcin, urinary calcium, and urinaryhydroxyproline, by bone biopsy with pathological assessment, and byassessment of family history. Examples of bone density techniquesinclude, for example, single- and dual photon absorptiometry,quantitative computed tomography, dual x-ray absorptiometry, andultrasonography. Preferred sites of analysis include the hip, wrist, andvertebrae. Other detection methods include low level x-ray on a fingeror wrist, ultrasound of the heel, and CT scan of the spine. In stillanother embodiment of the invention, a method of reducing bone tissuedamage associated with ROS includes administering an effective amount ofa composition of the present invention to an individual. The amount ofROS related damage by inhibits the production of reactive oxygenspecies.

In some embodiments, the ROS-mediated damage causes or exacerbates abone disease. Examples of such bone diseases include, but are notlimited to osteoporosis, including type I osteoporosis, type IIosteoporosis, age-related osteoporosis, disuse osteoporosis,diabetes-related osteoporosis, and steroid-related osteoporosis,periodontal disease, osteopenia, osteomalacia, osteolytic bone disease,primary and secondary hyperparathyroidism, multiple myeloma, metastaticcancers of the bone, for example, of the spine, pelvis, limbs, hip, andskull, osteomyelitis, osteoclerotic lesions, osteoblastic lesions,fractures, osteoarthritis, infective arthritis, ankylosing spondylitis,gout, fibrous dysplasia, and Paget's disease of the bone.

Recent work has indicated that these and other bone diseases may beexacerbated by ROS. ROS can have direct effects on various cells withinthe bones leading to apoptosis. Another possible mechanism by whichthese molecules can damage bone cells and tissue may be related to therole of ROS in bone resorption. For example, ROS produced by osteoclastsmay effectively suppress bone formation and bone healing. One skilled inthe art would be familiar with assays to determine various aspects ofbone healing. See, for example, Gadi Pelled, Ayelet Ben Arav, ColleenHock, David G Reynolds, Cemal Yazici, Yoram Zilberman, Zulma Gazit, HaniAwad, Dan Gazit, Edward Schwarz. Tissue Engineering Part A. Online Aheadof Editing: Jul. 21, 2009.

One embodiment of the invention relates to compositions and methods fortreating and/or preventing cellular and tissue damage caused by reactiveoxygen species released by osteoclasts in the process of boneresorption. In some embodiments, the compositions and methods of theinvention reduce ROS-mediated damage by inhibiting the production orrelease of ROS.

A variety of reactive oxygen metabolites (ROMs) are produced in themonovalent pathway of oxygen reduction. These ROMs are enzymaticallyproduced by osteoclasts and phagocytes such as monocytes andpolymorphonuclear neutrophils (PMNs) and frequently released in arespiratory burst. Neutrophils also produce ROMs constitutively. Theconstitutive production may contribute to ROS-mediated cellular damage.Hydrogen peroxide and other ROS play an important role in a host'simmunological defenses. Nevertheless, ROS produced in excessive amountsor at inappropriate times or locations, act to damage a host's cells andtissues, and thus can be detrimental to the host.

Examples of environmental and industrial toxins which cause damage tobone tissue include, without limitation, cigarette smoke, caffeine,alcohol, detergents, petroleum products, radiation, diethanoloamine,sodium laurel sulfate, propylene glycol, pesticides such as DDT andmirex, food additives and preservatives, heavy metals, organic solventssuch as formaldehyde and bromobenzene, and solvents such as dioxins,flurans, TCE, PCE, DCE, tetrachloroethylene, carbon tetrachloride, andvinyl chloride. As will be described in greater detail below, toxinsalso include many common drugs, such as steroids, chemotherapy drugs,hormones, and anticonvulsants. Damage to bone tissue results, at leastin part, by the detrimental release of ROS within a host or subject inresponse to such insults. Accordingly, compositions and methods fortreating damage to bone tissue caused by exposure to toxic substancesare provided. In one example, the administration of a ROS production andformation inhibiting composition of the present invention is useful forthe reduction in trauma to bone cells and tissues following exposure toindustrial and/or environmental toxins.

Numerous medications have been associated with damage to the bones. Suchdrugs include any substance or substances which act upon the bones tocause tissue damage. Examples of medications that have been associatedwith bone loss include, without limitation, corticosteroids, such asbetamethasone, budesonide, cortisone dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, and triamcinolone; cancertreatments, such as hormone therapy, including, for example, androgendeprivation in prostrate cancer, orchiectomy, hormone therapy such asreduced estrogen and/or progesterone for breast cancer or metastaticbreast cancer; thyroid hormone, such as thyroxine, for hyperthyroidism;anticonvulsants, such as barbituates, phenoarbital, phenyloin, andbenzodiazepines; and lupus and Crohn's disease treatments.

Accordingly, a composition of the present inventions can be administeredto an individual who is concurrently taking a drug or drugs which causetoxic side effects to mitigate bone loss caused by the drug. Theadministration of a composition of the invention is likewise useful forameliorating damage to bone tissue caused or exacerbated by bacterial,or viral infections. Staphylococcus aureus, Streptococcus pyogenes,Haemophilus influetazae, Myobacterium tuberculosis, salmonellas andcoliform bacteria, Pseudomonas aeruginosa, Treponema pallidum, andEscherichia coli are just a few examples of a species of pathogenicbacteria which invades the bones and causes tissue damage.

Accordingly, in one embodiment, a composition of the present inventionand methods for minimizing damage to bone tissue associated withbacterial, fungal, or viral infections are provided. A composition ofthe present invention may be administered alone or in combination withan antibiotic. The administration can be by either local or by systemicdelivery and any other suitable method known to one skilled in the art.Other methods of administration may also be suitable, such as oraladministration.

For oral administration, the composition of the present inventions canbe in powder form or incorporated into a tablet, aqueous or oilsuspension, dispersible powder or granule, microbead, emulsion, hard orsoft capsule, syrup or elixir. Administration of the composition of thepresent inventions can also be accomplished via parenteral deliverythrough subcutaneous, intravenous, intraperitoneal, or intramuscularinjection, intraocular, oral, transdermal, intranasal, or rectal and canutilize direct hypodermic or other injection or infusion means, or canbe mediated by a controlled release mechanism or any suitable methodknown to one skilled in the art.

The present invention also relates to any pharmaceutical,dermatological, medical, nutritional or cosmetic compositions comprisinga composition, fraction thereof or component obtained from eggshellmembrane using a process of the present invention. The composition mayinclude an effective amount of at least one or more components obtainedfrom solubilized eggshell membrane. In one aspect, the composition is ahydrolyzed, water-soluble protein composition from the eggshellmembrane, one of more fractions thereof such as a retentate fraction orpermeate fraction of components having the desired molecular mass (kDa),or individual components, e.g. proteins or polysaccharides, thereof. Thecomposition can be any dose or effective amount of the composition thatis safe and efficacious to achieve the desired result. As the diseases,disorders or conditions that would benefit from these compositions arewell known, the compositions may be designed such that they containappropriate levels effective for treatment of the particular disease,disorder or condition. The compositions may generally be used in anyformulation that is effective for treatment and the intended mode ofadministration. For example, compositions of elastin or collagen such asType I collagen, Type V collagen, and/or Type X collagen, for use indermatological, medical or cosmetic treatments may be formulated intopical, oral or injectable forms and the like. Compositions comprisingsolubilized proteins for use in nutritional or medical applications maybe formulated in any suitable form, e.g. aqueous or dried, andadministered by any effective route, such as orally or intramuscularly.As appreciated by one skilled in the art, compositions of the presentinvention can be administered in a variety of ways including oral,enteral, parenteral, topical, sublingual, by inhalation spray, rectaland other appropriate routes of administration, such as oral,subcutaneous, transdermal, transmucosal, iontophoretic, intravenous,intramuscular, intraperitoneal, intranasal, subdural, rectal, and thelike.

In other preferred embodiments, formulations of this invention arepharmaceutical compositions suitable for administration via variousroutes, preferably orally or topically, and for therapeutic and/orprophylactic administration.

A composition of the present invention, such as a pharmaceuticalcomposition, may comprise different types of acceptable carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it needs to be sterile for such routes ofadministration as injection. A composition of the present invention,such as a pharmaceutical composition, can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe foregoing as would be known to one of ordinary skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference). A number ofsuitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985) and in Dermatological Formulations:Percutaneous absorption, Barry (Ed.), Marcel Dekker Inc., 1983, bothincorporated herein by reference. Moreover, for a brief review ofmethods for drug delivery, see Langer, Science 249:1527-1533, 1990,which is also incorporated herein by reference.

The compositions or products of the invention can be applied to anyaffected part of the body. It will be appreciated that the presentmethods of treatment can be applied alone or in combination withtreatments for these conditions. The compositions useful in the presentmethods can be administered one time or multiple times, depending on thecomposition, the severity of the disease, disorder, or condition, andthe initial response of the condition to the treatment, for example,alleviating a symptom. For example, the compositions can be administered1, 2, 4, or more times per day, and can be administered every 1, 2, 4,7, or more days. Such treatments can be administered for a limitedduration, or indefinitely until the condition has resolved. Thecompositions can be applied locally as a “leave on” product, meaningthat the composition is applied to the individual and allowed to remainindefinitely at the site of application, or as a “wash off” product,meaning that the composition is allowed to remain at the site ofapplication for a limited amount of time, e.g., for a certain number ofseconds, minutes, hours, etc, and then removed.

Compositions containing solubilized components from eggshell membranesof the present invention may be in any form suitable for the intendedmode of administration, including, for example, a powder, a solution, asuspension, or an emulsion. Liquid carriers are typically used inpreparing solutions, suspensions, and emulsions. Liquid carrierscontemplated for use in the practice of the present invention include,for example, water, saline, pharmaceutically acceptable organicsolvent(s), pharmaceutically acceptable oils or fats, and the like, aswell as mixtures of two or more thereof. The liquid carrier may containother suitable pharmaceutically acceptable additives such assolubilizers, emulsifiers, nutrients, buffers, preservatives, suspendingagents, thickening agents, viscosity regulators, stabilizers, and thelike. Suitable organic solvents include, for example, monohydricalcohols, such as ethanol, and polyhydric alcohols, such as glycols.Suitable oils include, for example, soybean oil, coconut oil, olive oil,safflower oil, cottonseed oil, and the like. For parenteraladministration, the carrier can also be an oily ester such as ethyloleate, isopropyl myristate, and the like. Compositions of the presentinvention may also be in the form of microparticles, microcapsules,liposomal encapsulates, and the like, as well as combinations of any twoor more thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-propanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents. The compositions of the presentinvention may be administered orally, parenterally, sublingually, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional nontoxic pharmaceutically acceptable orphysiologically acceptable carriers, adjuvants, and vehicles as desired.Topical administration may also involve the use of emulsions, creams,ointments, transdermal patches or ionophoresis devices. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection, or infusion techniques.

As described elsewhere herein, the compositions useful in the presentmethods can be administered to an individual using a variety of routes,such as oral or parenteral, and formulations. One skilled in the artwill be well versed in selecting the appropriate formulation and routein which to administer a composition of the invention to an individual,such as a human, mammal, animal, companion animals or farm animals. Insome cases, any of the present compositions are administered to anindividual at the local site of the disease, disorder or condition,whether by local injection, topical administration, or any othersuitable method. As such, administration of the compositions can beachieved in various ways, including by topical application of thecomposition to the site of the disease or condition, i.e., directapplication of a formulation to the affected skin. If desired,compositions can be formulated for injection and injected locally at thesite of the disease or condition, e.g., local subcutaneous injection atthe site of the disease, for example, to treat a joint disorder.

For any composition used in a method of the invention or product of theinvention, a therapeutically effective amount or dose can be estimatedinitially in vitro or in vivo data. Initial dosages can also beformulated by comparing the effectiveness of the compounds used in themethods of the present invention in model assays with the effectivenessof known compounds. One having ordinary skill in the art could readilydetermine an effective dosage in humans or other mammals and animals.

Dosage amount and interval may be adjusted individually to providelevels of the composition which are sufficient to achieve and/ormaintain their desired effect. One having skill in the art will be ableto optimize therapeutically or prophylactically effective amounts ordosages without undue experimentation.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXAMPLE

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. The disclosure of each reference set forthherein is incorporated herein by reference in its entirety.

Example 1

Shown below is data resulting from the analysis of various samples ofthe resulting protein composition obtained by solubilizing avianeggshell membrane using a process of the present invention.

As indicated below, the solubilized protein composition was spray dried(SD), freeze dried (FD), or paddle dried (PD). Paddle dried is a commontechnique used by some egg breaking facilities to dry eggshells on acommercial scale.

The total protein (TP) concentration of the solubilized proteincomposition was measured using Leco instruments (St Joseph, Mich.)following the Association of Analytical Chemists (AOAC) protein bycombustion method 990.03. The solubilized protein composition wasdetermined “as is” in its current physical state as well as on a “drybasis”.

The ash content was determined using AOAC method 942.05. The percentsolubility of the resulting protein composition was determined usingstandard techniques. The percent collagen, elastin and sulfated glycanswere determined using various commercially available assays, forexample, calorimetric kits such as Sircol, Fastin, Blyscan assays(Biocolors Ltd, Northern Ireland). Percent hyaluronic acid wasdetermined by measuring uronic acid by a carbazole method. The aminoacid profile was performed by Eurofins Scientific, Inc. (Des Moines,Iowa). A Standard Plate Count (SPC) procedure was used to determine thepresence of bacteria in each of the samples, as indicated below by SPC.

The measurement of color was determined using the Hunter L, a, bstandard color scale, which is described below. Hunter L, a, b valuesare standard color scale values that indicate differences in brightness,hue and saturation using a standard color system which relates lightnessas L values, and hue and croma as a combination of a and b values on acoordinate scale, where a represents redness-greenness and b representsyellowness-blueness. L values describe the degree of darkness, where avalue of 100 equals white and that of 0 equals black. a-values describethe degree of redness, which increases with an increasing a-value.b-values describe the degree of yellowness, which increases withincreasing b-value. L, a, b and opacity theory and measurement arefurther described in the Hunter Lab Instruction Manual Hunter. L, a, band color scale values and opacity may be measured using a calorimeteravailable from Hunter Associate Laboratory, Inc. of Reston, Va., U.S.A.or the Color Machine Model 8900 available from Pacific Scientific.

As shown in FIG. 2, wet, dry or frozen membranes were put into astainless (316) tank and 12% NaOH was added and the mixture wasincubated at 32-40° C. overnight. After hydrolysis the mixture wascooled to 38-45° F. to slow continued hydrolysis. The mixture wascentrifuged to separate eggshells from solubilized proteins. Thecentrifuged solubilized proteins were then dialyzed through 3,000 MWCOmembranes until ash was reduced to a conductivity reading of 2-4 mS/cm.The proteins were then pH adjusted with 12% acetic acid to a pH of6.8-8. The pH adjusted protein mixture was then concentrated to a solidscontent of 25% to 30% at which time it could be spray dried or freezedried. Tables 1 and 2 below show the percent analysis of samples fromthe resulting composition.

TABLE 1 Percent analysis of sample compositions. Type (SD, Leco TP Ash %FD, Leco TP % dry Ash % dry Sample PD) % as-is basis as-is basis Moist %Solids % Solubility % Collagen % 1 FD 90.45 94.50 6.67 6.97 4.29 95.7195.40 16.96 2 FD 91.56 95.64 6.96 7.27 4.27 95.73 92.26 18.49 3 FD 90.2592.61 7.35 7.54 2.55 97.45 99.70 17.62 4 SD 91.66 100.03 6.22 6.79 8.3791.63 98.16 15.44

TABLE 2 Percent analysis of sample compositions. Hyaluronic ElastinSulfated SPC's Salmonella Color Sample Acid % % Glycans % (cfu's/g)(neg/25 g) (L) Color (a) Color (b) 1 0.13 29.52 ND <100 neg 63.41 2.4118.88 2 0.17 24.20 ND <100 neg 63.78 3.35 20.64 3 0.15 22.50 0.01 1,800neg 57.47 0.57 15.42 4 0.20 24.52 ND <1,000 neg 86.37 −0.48 11.08

As shown in FIG. 3, various soluble protein compositions may be obtainedusing various membrane sizes in accordance with the methods of thepresent invention. A typical test analysis of the compositions obtainedfrom processing 600 pounds of eggshell membranes obtained from eggs fromchicken (Pullus) using various sized membranes are shown in Tables 3-6.

Example 2 Process for Making the Product Referred to as Composition a inFIG. 3A

The process for making the product referred to as Composition A includesadding eggshell membranes (wet, dry or frozen) to a jacketed 316stainless tank followed by 5% sodium hydroxide. The tank contents wereheated to 50° C. while stirring until hydrolysis was completed which is3 to 4 hours. The tank stirrer was shut off after hydrolysis wascomplete and eggshell is allowed to settle out. (Approx. 30 min.)Hydrolysis was monitored until no membrane was visible and only theeggshell remains.

The hydrolyzed membrane was pumped through a 250-500 μm membrane andinto the centrifuge for separation of eggshell fines from thesupernatant liquid.

After centrifuging, the supernatant liquid was pumped into a 1,000Dalton to 10,000 Dalton membrane system, where the ash was removed untilit measures less than 4 milliSiemens/cm at which time the pH wasadjusted to pH 6.8-7.6 with 0.5% acetic acid and dialyzed with one morevolume of water. The dialyzed, pH adjusted supernatant was concentratedto 20-30% solids and then spray dried in a nozzle dryer to generateComposition A. FIG. 3A and Table 3 below shows the resultingcomposition.

TABLE 3 Percent analysis of components of Composition A. Composition AAnalysis % Total protein 92.59 Ash 6.51 Moisture 5.00 Solids 95.01Collagen 32.17 Hyaluronic Acid 1.59 Elastin 21.62

Further analytical results indicate that the following amino acids arepresent in the following Composition A:

TABLE 4 Percent analysis of amino acid present in the Composition A.Amino Acid (%) Tryptophan 3.53 Cystine 2.47 Methionine 3.79 AsparticAcid 8.33 Threonine 3.08 Serine 3.30 Glutamic Acid 14.61 Proline 9.85Glycine 4.38 Alanine 2.25 Valine 7.51 Isoleucine 3.47 Leucine 4.72Tyrosine 1.72 Phenylalanine 1.57 Total Lysine 5.99 Histidine 3.23Arginine 6.58 Hydroxyproline 0.22

Example 3 Process for Making the Product Referred to as Composition B inFIG. 3A

Egg membranes (wet, dry or frozen) were added to a jacketed 316stainless tank followed by 5% sodium hydroxide. The tank contents wereheated to 50° C. while stirring until hydrolysis was completed which is3 to 4 hours. The tank stirrer was shut off after hydrolysis wascomplete and eggshell is allowed to settle out. (Approx. 30 min.)Hydrolysis was monitored until no membrane is visible and only theeggshell remains.

The hydrolyzed membrane is pumped through a 250-500 μm membrane and intothe centrifuge for separation of eggshell fines from the supernatantliquid.

After centrifuging, the hydrolyzed membrane was diafiltered through a100,000 molecular weight cutoff membrane. The retentate was saved andconductivity was reduced from 140 milliSiemens/cm to 4 milliSiemens/cmand concentrated to 25-30% solids and spray dried. The results are shownin the following table and in FIG. 3A.

TABLE 5 Percent analysis of components of the Composition B composition.Composition B Analysis % Total protein 94.10 Ash 6.67 Moisture 4.27Solids 95.73 Collagen 23.70 Hyaluronic Acid 1.27 Elastin 37.95

Example 4 Process for Making the Product Referred to as Composition C inFIG. 3B

The permeate was collected from the 100,000 molecular weight cutoffmembranes and dialyzed with 10,000 molecular weight membranes.Conductivity was reduced from 140 milliSiemens/cm to 4 milliSiemens/cmand concentrated to 25-30% solids and spray dried. The resultingcomposition is provided in FIG. 3B and Table 6 below.

TABLE 6 Percent analysis of components of the Composition C composition.Composition C Analysis % Total protein 90.18 Ash 8.09 Moisture 5.14Solids 94.86 Collagen 15.93 Hyaluronic Acid 2.00 Elastin 2.35

Example 5

Process for making BIO1 and BIO2 products and composition analysis.

BIO1

As used herein, BIO1 refers to a an eggshell membrane compositionobtained as a 3 kDa permeate using a 3 kDa membrane and nanofilter togenerate a nanofilter retentate using the methods described herein.SKU313 and 314 refer to a spray dried form of BIO1 and freeze dried formof BIO1 respectively. The Bio1 composition has molecules and componentslower than 3 kDa, but higher than 450 Da.

The process for making the product referred to as BIO1 includes addingeggshell membranes (wet, dry or frozen) to a jacketed 316 stainless tankfollowed by 5% sodium hydroxide. The tank contents were heated to 50° C.while stirring until hydrolysis was completed which is 3 to 4 hours. Thetank stirrer was shut off after hydrolysis was complete and eggshell isallowed to settle out. (Approx. 30 min.) Hydrolysis was monitored untilno membrane was visible and only the eggshell remains.

The hydrolyzed membrane was pumped through a 250-500 μm membrane andinto the centrifuge for separation of eggshell fines from thesupernatant liquid.

After centrifuging, the supernatant liquid was pumped into a 1,000Dalton to 3,000 Dalton membrane system, where the ash was removed untilit measures less than 4 milliSiemens/cm at which time the pH wasadjusted to pH 6.8-7.6 with 1.2% acetic acid and dialyzed with one morevolume of water. The permeate passing through the 3 Kda membrane wassubjected to a nanofilter and the NF retentate was dialyzed, pH adjustedsupernatant was concentrated to 20-30% solids and then spray dried in anozzle dryer to generate BIO1 or freeze dried. The table below shows theresulting composition.

BIO2

As used herein, BIO2 refers to a an eggshell membrane compositionobtained as a 3 kDa retentate using a 3 kDa membrane using the methodsdescribed herein. SKU309, BiOvacore, Ovacore, Biova Flex, OvaFlex,BiovaDerm and Ovaderm all refer to a spray dried form of BIO2 and theterms are used interchangeably herein. The Bio2 composition hasmolecules and components higher than 3 kDa.

The process for making the product referred to as BIO2 includes addingeggshell membranes (wet, dry or frozen) to a jacketed 316 stainless tankfollowed by 5% sodium hydroxide. The tank contents were heated to 50° C.while stirring until hydrolysis was completed which is 3 to 4 hours. Thetank stirrer was shut off after hydrolysis was complete and eggshell isallowed to settle out. (Approx. 30 min.) Hydrolysis was monitored untilno membrane was visible and only the eggshell remains.

The hydrolyzed membrane was pumped through a 250-500 μm membrane andinto the centrifuge for separation of eggshell fines from thesupernatant liquid.

After centrifuging, the supernatant liquid was pumped into a 1,000Dalton to 3,000 Dalton membrane system, where the ash was removed untilit measures less than 4 milliSiemens/cm at which time the pH wasadjusted to pH 6.8-7.6 with 1.2% acetic acid and dialyzed with one morevolume of water. The 3 kDa retentate was dialyzed, pH adjustedsupernatant was concentrated to 20-30% solids and then spray dried in anozzle dryer to generate BIO2. The table below shows the resultingcomposition.

Test Result-SKU309 Result-SKU313 Total Protein 91.90% 83.71% Ash 7.39%16.40% Moisture 4.18% 4.09% Collagen 25.85% 3.07% Hyaluronic Acid 1.63%3.78% Elastin 22.11% negligible Chondroitins 0.03% none detectedGlucosamine 0.82% 0.44% ORAC_(hydro) 423 μmoleTE/g 829 μmoleTE/g AerobicPlate Count <10,000 cfu/g <10 cfu/g Salmonella negative/25 g negative/25g Coliforms <10 MPN/g <3 MPN/g E. coli <10 MPN/g <3 MPN/g B. cereus <3MPN/g <3 MPN/g Yeast <10 cfu/g <10 MPN/g Mold <10 cfu/g <10 MPN/g S.aureus <10 cfu/g not tested Arsenic 0.03 ppm <0.02 ppm Lead 0.05 ppm0.02 Cadmium <0.01 ppm <0.01 Mercury <0.01 ppm <0.01 Bulk Density 0.22g/cc not tested Calcium 0.35% not tested Water Activity 0.1 not tested

Amino Acid Analysis:

SKU # Amino Acid (%): 309 314 Tryptophan 4.12 2.37 Cystine 3.00 1.91Methionine 4.18 3.56 Aspartic Acid 8.16 8.34 Threonine 2.94 2.40 Serine3.06 3.54 Glutamic Acid 12.02 12.62 Proline 8.87 8.04 Glycine 4.91 7.41Alanine 2.30 3.06 Valine 7.06 5.78 Isoleucine 3.30 2.72 Leucine 4.484.56 Tyrosine 1.88 1.53 Phenylalanine 1.65 1.46 Total Lysine 6.07 0.63Histidine 3.37 3.01 Arginine 5.63 4.28 Hydroxyproline 0.33 1.50 LysineAvailable 4.71 0.63 Lysine Unavailable 1.37 0.55 Aspartic Acid (Free)<0.01 0.08 Threonine (Free) <0.01 0.14 Serine (Free) <0.01 <0.01Glutamic acid (Free) <0.01 0.02 Proline (Free) <0.01 0.32 Glycine (Free)<0.01 <0.01 Alanine (Free) <0.01 0.01 Cystine (Free) <0.01 0.06 Valine(Free) <0.01 <0.01 Methionine (Free) <0.01 0.04 Isoleucine (Free) <0.010.08 Leucine (Free) <0.01 <0.01 Tyrosine (Free) <0.01 0.16 Phenylalanine(Free) <0.01 <0.1 Lysine (Free) <0.01 <0.01 Histidine (Free) <0.01 <0.01Arginine (Free) <0.01 <0.01

Example 6 Lipoxygensase Inhibition

Lipoxygenase. is an enzyme involved in inflammation. A lipoxygenaseinhibitor screening kit was obtained from Cayman Chemical, and usedaccording to the guidelines of the manufacturer. In this assay, apurified 15-Lipoxygenase enzyme from soybean was mixed with thesubstrate arachidonic acid in the absence versus presence of each of theBIO1 and BIO2 fractions. The hydroxyperoxides that are produced as aresult of the lipoxygenase reaction generated a colored product and thelight absorbance was measured. FIG. 4 shows the dose-dependentinhibition of hydroxyperoxide formation by BIO1 versus BIO2. Bothcomplementary fractions show activity.

As shown in FIG. 4, in the lipoxygenase inhibition assay, both productsdid well but BIO1 (1:10) had a 68% inhibition in which the data washighly statistically significant (P<0.0009). BIO2 (1:10) was stillcapable of inhibiting lipoxygenase by 12% at the highest dose tested;this data was highly statistically significant (P<0.0003).

Both the low-molecular weight composition of BIO1 and high-molecularweight composition of BIO2 fractions of eggshell membrane (ESM) containscompounds able to inhibit the generation of reactive oxygen species bythe enzyme lipoxygenase.

Both the low-molecular weight composition of BIO1 and high-molecularweight composition of BIO2 fractions of ESM contained compounds able tointerfere with the generation of hydroxyperoxyl free radicals generatedby the enzymatic function of lipoxygenase.

Example 7 Inhibition of the Formation of ROS

The BIO1 and BIO2 fractions from ESM contains compounds able to inhibitthe formation of Reactive Oxygen Species (ROS) by humanpolymorphonuclear (PMN) cells.

Freshly purified human polymorph nucleated cells (PMN) were used fortesting ROS formation. This cell type constitutes approximately 70% ofthe white blood cells in humans. PMN produce high amounts of ROS uponcertain inflammatory stimuli. PMN cells are complex and capable ofreacting in several ways upon exposure to natural products as follows:

-   -   1. Passive absorption of antioxidants into the cells, leading to        neutralization of ROS within the cells;    -   2. Active signaling by compounds in the natural product (for        example glucans) leading to increased ROS production;    -   3. Active signaling by compounds in the natural product leading        to a reduced inflammatory response by the cell and therefore a        reduced production of ROS.

When PMN cells are exposed to a natural product, it is possible that anincrease in spontaneous ROS formation may occur, if the productpossesses immune activating properties, including a stimulation of PMNcell anti-bacterial activity. Alternatively, a decrease may be observed,which means the natural product reduced the PMN cell spontaneous ROSformation.

PMN cells were isolated by applying heparinized peripheral venous bloodon top of a double-gradient of 3 ml Histopaque 1119 and 3 mL ofHistopaque 1077. This was centrifuged for 25 minutes at 2400 rpm. Theplasma and PBMC was removed, and the PMN fraction harvested. The PMNcells were washed twice in phosphate-buffered saline without calcium ormagnesium, and resuspended in RPMI 1640. Serial dilutions of BIO1 andBIO2 were added to PMN cells and the cells were incubated for 20minutes. Subsequently, cells were washed twice to remove the ESMfractions, and the precursor dye DCF-DA was added. The PMN cells wereincubated with DCF-DA for 1 hour, after which time unabsorbed DCF-DA wasremoved by two washes. Oxidative burst was then induced, either byadding H₂O₂ or by adding the bacterial peptide f-MLP for 45 minutes.Cells were washed, resuspended in RPMI 1640, and kept on ice untilsamples were acquired by flow cytometry. Oxidative damage, such as whathappens during a reactive oxidative burst in PMN cells, transforms theDCF-DA precursor dye into a fluorescent dye. The fluorescence intensityof the cells is a measure of the intensity of the oxidative burst.

Freshly purified human PMN were exposed to the test products. During theincubation with the test products, any antioxidant compounds able tocross the cell membrane can enter the interior of the PMN cells. Thenthe cells were washed, loaded with the DCF-DA dye, which turnsfluorescent upon exposure to reactive oxygen species. Oxidation istriggered by addition of H₂O₂. The fluorescence intensity of the PMNcells was evaluated by flow cytometry. The low fluorescence intensity ofuntreated control cells served as a baseline and PMN cells treated withH₂O₂ alone serve as a positive control. If the fluorescence intensity ofPMN cells exposed to the extract, and subsequently exposed to H₂O₂, isreduced compared to H₂O₂ alone, this indicates that the test product hasanti-inflammatory effects. In contrast, if the fluorescence intensity ofPMN cells exposed to a test product is increased compared to H₂O₂ alone,this indicates that a test product has pro-inflammatory effects.

FIG. 5 shows that both products inhibited formation of ROS by PMN cells,both under normal culture conditions and under oxidative stress. Asshown in FIG. 5A, In this case both products were capable at helpingreduce the baseline level of ROS. BIO1 (1:10) helped reduce spontaneousROS formation by 58%, this data was highly significant (P<0.0000005).BIO2 (1:10̂3) was also capable at reducing spontaneous ROS formation by46% and this data was highly statistically significant (P<0.000002).FIG. 5B illustrates the amount of protection a natural product canprovide when a PMN cell has been exposed to oxidative stress, in thiscase by being treated with H₂O₂. In this case both products were capableat inhibiting ROS formation under conditions of oxidative stress acrossall dilutions tested. Both BIO1 (1:10) and BIO2 (1:10) reduced ROSformation by 37%; BIO1 data was highly statistically significant(P<0.000003) and BIO2 data was also highly statistically significant(P<0.00002).

Example 8 Migratory Behavior 3. Effect on PMN Cell Chemotactic MigrationTowards Three Chemo-Attractants.

The PMN cell is a highly active and migratory cell type. The migratorybehavior of this cell type is divided into at least two types:

a) random migration and

b) directed migration.

Random migration is part of normal immune surveillance, whereas directedmigration happens towards specific chemoattractants.

We have established a migration test where both types of migration aretested in parallel. Furthermore, the directed migration is testedtowards three distinctly different chemotactic compounds:

i) Bacterial peptide f-Met-Leu-Phe;

ii) The inflammatory cytokine Interleukin-8 (IL-8); and

iii) Leukotriene B4 (LTB4).

The migration towards f-MLP is a reflection of anti-bacterial immunedefense mechanisms, whereas the migration towards IL-8 and LTB4 reflectsmechanisms involved in the escalation of the inflammatory response.

We have found interesting evidence during evaluation of several naturalproducts that some test products may specifically reduce directed PMNmigration towards the inflammatory mediators IL-8 and/or LTB4 whileallowing PMN migration towards bacterial peptide as part of the normalanti-bacterial immune defense.

The differential effect on PMN cell random migration as well aschemotactic migration towards three different inflammatorychemo-attractants: bacterial fmlp, IL-8, and Leukotriene B4 were tested.

Enhancement of PMN Cell Random Migratory Behavior

The migration of polymorphonuclear (PMN) cells was tested using dualchamber 96-well migration plates. PMN cells are plated in the topchambers. A filter separates the two chambers, and 3 micron pores allowmigration of cells from top to bottom chamber. This in vitro assay isdesigned to mimic the migration of cells from blood (top chamber) intotissue (bottom chamber).

PMN cells were plated in the top chambers with and without BIO1. Controlwells included cells un-exposed to product and without chemoattractantin the bottom wells. The random migration activity of PMN cells resultedin measurable amounts of PMN cells in the bottom chambers. The relativeamount of cells was determined by staining of the cells in the bottomchambers using the CyQuant fluorescent probe. When BIO1 was added to PMNcells in the top chambers, the random migratory activity of the PMNcells was increased. BIO2 had a lesser effect.

Freshly purified PMN cells were set up in cultures in double-chambermigration plates, where the bottom chamber mimics tissue, and the topchamber mimics the blood stream. Cells are plated in the top chamberswith and without test product, and the chemo-attractants will be presentin the bottom chambers. Control wells included cells un-exposed to testproducts and without chemo-attractant in the bottom wells, and allowevaluation of baseline random migration. All assays were performed intriplicates, and repeated 3 times.

Inhibition of PMN Cell Migration Towards the Inflammatory MediatorLeukotriene B4

The migration of polymorphonuclear (PMN) cells was tested using dualchamber 96-well migration plates. PMN cells are plated in the topchambers and different chemotactic agents can be added to the bottomchamber. A filter separates the two chambers, and 3 micron pores allowmigration of cells from top to bottom chamber. This in vitro assay isdesigned to mimic the migration of inflammatory cells from blood (topchamber) into tissue (bottom chamber), with inflammatory mediators aschemoattractants.

PMN cells were plated in the top chambers with and without serialdilutions of BIO1 or BIO2 and the inflammatory chemo-attractantLeukotriene B4 (LTB4) was present in the bottom chambers. Control wellsincluded cells un-exposed to product and without chemoattractant in thebottom wells. The directed migration of PMN cells resulted in measurableamounts of PMN cells in the bottom chambers. The relative amount ofcells was determined by staining of the cells in the bottom chambersusing the CyQuant fluorescent probe. When BIO1 was added to PMN cells inthe top chambers, the migration of the PMN cells towards LTB4 in thebottom chambers was reduced. BIO2 had a lesser effect.

FIGS. 6A and B show that both products were capable at increasing randommigration cellular behavior linked to normal immune surveillance. BIO1(1:10) increased immune surveillance by 81% and was highly statisticallysignificant (P<0.000003). BIO2 (1:10) increased immune surveillance by52% and was also statistically significant (P<0.008).

Higher doses of BIO1 and BIO2 increased migration towards the bacterialpeptide f-MLP (FIG. 6F) implicating that an anti-microbial response wastriggered. BIO1 (1:10) increased migration by 59% and this data wasstatistically significant (p<0.002). BIO2 (10̂2) also increased migrationby 67% and was highly statistically significant (p<0.000006).Surprisingly, the more BIO2 was diluted a decrease in migration wasobserved.

BIO1 treatment of PMN cells resulted in a mild increase of PMN cellmigration towards IL-8. BIO1 (10̂3) increased migration by 27% and thedata was statistically significant (P<0.01). For BIO2 there were twodifferent and interesting effects. At the highest dose of BIO2 tested,an increased migration was seen. However, when BIO2 is further dilutedan anti-inflammatory effect was observed, as BIO2 treatment of PMN cellsresulted in a reduction in the migration towards LTB4. BIO2 (10̂4)decreased migration by 69% and was highly significant (P<0.0002). SeeFIG. 6E.

BIO2 was capable at reducing migration across all dilution sets and thestrongest reaction (10̂4) was shown to reduce migration by 72%, the datawas statistically significant (P<0.02). BIO1 (10̂3) was not able toreduce migration but did increase migratory activity by 55%; data wasnot significant. See FIGS. 6C and 6D.

Discussion:

-   -   Lipoxygenase inhibition: BIO1 strongly inhibited Lipoxygenase        enzymatic activity, whereas BIO2 mildly inhibited Lipoxygenase        enzymatic activity.    -   Inhibition of ROS formation: Both products performed equally        well in inhibiting oxidative stress-induced ROS formation.    -   PMN cell migration:        -   BIO1 activated several aspects of PMN cell migratory            behavior.        -   Both BIO1 and BIO2 elicited strong anti-microbial migratory            behavior in PMN cells.        -   BIO2 showed the best data in terms of inhibiting PMN cell            migration in response to the inflammatory mediators IL-8 and            LTB4, with a particular strong and clear inhibition of            LTB4-mediated migration.

Example 9

Evaluation of protection of cellular viability (protection fromprogrammed cell death (apoptosis)) in the absence versus presence ofoxidative stress.

Oxidative damage can trigger premature cellular death by a mechanismcalled apoptosis (programmed cell death). This death pathway can bemonitored by highly specific cellular markers. Protection from apoptosiscan be monitored as delay or absence of these markers.

Apoptosis is a carefully regulated process of cell death that occurs asa normal part of cellular development. In contrast to necrosis, a formof cell death resulting from acute cellular injury, apoptosis is carriedout in an ordered process.

The human vascular anticoagulant, annexin V, is a Ca2+-dependentphospholipid-binding protein that has a high affinity forphosphatidylserine (PS). In normal viable cells, phosphatidylserine islocated on the cytoplasmic surface of the cell membrane. However, inapoptotic cells, PS is translocated from the inner to the outer leafletof the plasma membrane, thus exposing PS to the external cellularenvironment. Annexin V labeled with a fluorophore can identify apoptoticcells by binding to PS exposed on the outer leaflet. AnnexinV-FITC wasused to label apoptotic cells. Co-staining with 7AAD, which only stainscells at a late phase of cell death where the cell membrane iscompromised, allows analysis to distinguish early and late apoptosis.Cells staining only with 7AAD, without Annexin V, reflect necroticcells.

Among freshly isolated human peripheral blood mononuclear cells, aproportion is already on an apoptotic path. When cultured in vitro,these cells will continue the apoptotic process. Early apoptosis isreversible. By culturing freshly purified human lymphocytes with andwithout test product, and then staining for apoptotic cells, we werecomparing the ability of the test product to rescue cells already on thepath to apoptosis.

In parallel, H₂O₂ was added to trigger oxidative stress-inducedapoptosis, and assess whether the test product was able to protect theviability of cells that were under severe oxidative stress.

The testing was performed where each testing condition, including eachserial dilution of test product, was performed in triplicate. Theexperiment was repeated three times using PBMC from different blooddonors. Samples were stored dark and acquired by flow cytometry within 4hours. Acquisition was performed using FACSCALIBUR™ flow cytometer andCELL-QUEST™ software. Analysis of fluorescence intensity of the markerswas performed using FLOWJO™ software.

Results: Evaluation of protection of cellular viability (protection fromprogrammed cell death (apoptosis)) in the absence versus presence ofoxidative stress.

When examining the effects of cellviability/death of an overnightincubation, six parameters were analyzed:

1. Percentage of viable cells under normal cell culture (FIG. 8A);

2. Percentage of viable cells under oxidative stress (FIG. 9A);

3. Percentage of total apoptosis under normal cell culture (FIG. 8B);

4. Percentage of total apoptosis under oxidative stress (FIG. 9B);

5. Percentage of necrotic cells under normal cell culture (FIG. 8C);

6. Percentage of necrotic cells under oxidative stress (FIG. 9C).

Viable cells (UT) are the percent of cells that exhibit very lowstaining for both Annexin V and 7AAD after an overnight incubationwithout product. This value is compared to cells that have beenincubated overnight with serial dilutions of product.

In the absence of H₂O₂ treatment, both BIO1 and BIO2 increased thepercentage of viable cells following overnight incubation. These mildchanges were not statistically significant.

The decrease in percent viable cells seen with the highest doses of BIO1and BIO2 (10 g/L) most likely reflect adverse effects of both productson cell viability at this high a concentration.

Under oxidative stress, BIO1 (1 g/L) increased the percent of viablecells by up to 30% above the untreated (UT) negative control samples.This increase was statistically significant (P<0.03 for 1 g/L). Whencells were placed under the same oxidative stress but now in thepresence of BIO2 (0.1 g/L), an increase of 15% viable cells was seen,however the data was not statistically significant. Once again at 10g/L, BIO2 had an adverse effect on cell viability.

Total apoptosis is the combination of all cells involved in earlyapoptosis and late apoptosis. When cells were treated with BIO1overnight in the absence of H₂O₂, BIO1 (0.1 g/L) reduced the amount oftotal apoptosis by 14% below that of the untreated (UT) negative controlsamples, however the data was not statistically significant. BIO2 (0.1g/L) also reduced the amount of total apoptotic cells, however, thisreduction was not statistically significant. At the 10 g/L dose,overnight treatment with BIO2 resulted in an increase in totalapoptosis.

Under oxidative stress BIO1 (1 g/L) reduced the percent of totalapoptotic cells by up to 9% below that of the untreated (UT) controlsamples (at 1 g/L), however the data was not statistically significant.The highest dose of BIO1 (10 g/L) increased the percentage of cellsundergoing apoptosis in response to oxidative stress. BIO2 did notrescue cells from apoptosis under oxidative stress but rather had anadverse effect on cell viability at the 2 highest doses tested.

When cells were incubated overnight with BIO1 (0.1 g/L) necrotic cellswere reduced by 10% below that of the untreated control samples, howeverthis small change was not statistically significant. BIO2 at the 1 g/Ldose reduced the percent of necrotic cells by 32%. This reduction onlyreached borderline statistical significance (P<0.07). At the highestdose tested (10 g/L), both products increased cell necrosis.

Cells undergoing oxidative stress induced by treatment with H₂O₂, wereprotected from necrosis in the presence of BIO1 in a cleardose-dependent manner. At 10 g/L BIO1 treatment resulted in a highlystatistically significant reduction of 47% (P<0.0006).

When cells were treated with BIO2 (1 g/L) followed by exposure to H₂O₂ a27% reduction in percent necrotic cells was observed and wasstatistically significant (P<0.005 at 1 g/L). The highest dose of BIO2tested (10 g/L) had an adverse effect on cells leading to increasednecrosis.

Under conditions of oxidative stress, BIO1 increased the percent ofviable cells above the untreated (UT) negative control samples, both byreducing the percent of cells in apoptosis and of cells in necrosis.BIO2 also reduced the amount of total apoptotic and necrotic cells atsome doses tested on cells.

Example 10 Protection of Mitochondrial Function Under Conditions ofOxidative Stress

Mitochondria are the intracellular organelles responsible for producingcellular energy. Decreased mitochondrial function has been associatedwith ageing, inflammatory conditions, and degenerative illnesses. Thisassay measures mitochondrial function, and examines whether a testproduct contains compounds capable of supporting healthy mitochondrialfunction, as well as protecting mitochondrial function in cells underoxidative stress.

Freshly purified human peripheral blood mononuclear cells (PBMC) werecultured in the absence versus presence of serial dilutions of testproduct, either without further treatment or in the presence ofoxidative stress. After incubation, the level of mitochondrial functionwas measured by staining with the marker MitoTracker-Red, which becomesfluorescent as a result of, and in proportion to, mitochondrialfunction. Therefore, the resulting fluorescence intensity wasproportional to mitochondrial function, and was evaluated by flowcytometry. A decrease in fluorescence intensity reflects a reduction ofmitochondrial function, as mitochondria were damaged by oxidativestress. If the presence of test product protects mitochondrial function,then higher fluorescence intensities will be observed.

Mitochondrial function is measured through the use of amitochondria-specific fluorescent dye. The mean fluorescence intensity(MFI) of the MitoTracker-Red dye reflects the number and functionalityof mitochondria within a cell. When a cell is producing moremitochondria or the activity of mitochondria within the cell increases,an increase in the mean fluorescence will be seen.

FIG. 7 shows that when cells were treated overnight with BIO1 (10 g/L)an increase in mitochondrial function was observed. BIO2 also had aneffect, even thought to a lesser degree. The protective effect was seenboth under normal culture conditions and under conditions of oxidativestress.

Evaluation of protection of mitochondrial function in the absence versuspresence of oxidative stress.

When cells were treated overnight with BIO1 (10 g/L) a 17% increase inmitochondrial function was observed (P<0.01). When BIO2 (10 g/L) wasadded to cells in an overnight incubation, mitochondrial function wasincreased by 6% above untreated cells, however the data was notstatistically significant. See FIG. 7A.

When cells were cultured in the presence of product and H₂O₂ it wasobserved that BIO1 (1 g/L) increased mitochondrial function by 25% abovecells treated with H₂O₂ in the absence of product. This increase wasstatistically significant (P<0.03). Under the same conditions, treatmentwith BIO2 (1 g/L) increased mitochondrial function by 6%, however thedata was not statistically significant. See FIG. 7B.

Example 11

Anti-wrinkle effect of the BiOvaDerm™ (BIO2, 309) fractions in vivo. Theeffects of ESM fractions on facial wrinkles was measured.

A single-site open-label Pilot Study was conducted to evaluate theeffectiveness of BiovaDerm™ in reducing aged skin related wrinkles andits impact on sun damaged skin. The composition described in this studyis a topical skin cream, which was formulated with 10% of BiOvaDerm™(Bio2) by weight suspended in a base cream “carrier” to facilitateapplication. Twenty three (23) subjects were enrolled and evaluated inthree groups that differed in the length of time they followed a dailytreatment regime:

-   -   Group 1 consisted of six (6) subjects that were instructed to        apply study product for one (1) week. Group 1 used the cream        daily for one week after which they were asked to return the        cream and continue their normal regime observing the exclusion        criteria. Subject evaluations were conducted at initial visit,        at the end of week 1. Subject evaluations continued to        facilitate capture of “rebound” data for 4 weeks after product        application had ceased.    -   Group 2 consisted of nine (9) subjects and they were instructed        to apply study product for two (2) weeks. Group 2 used the cream        daily for two weeks after which they were asked to return the        cream and continue their normal regime observing the exclusion        criteria. Subject evaluations were conducted at initial visit,        week 2 and weeks 4 and 6. This again facilitated capture of        “rebound” data four weeks after product application had ceased.    -   Group 3 contained eight (8) subjects and they were instructed to        apply study product for four (4) weeks. Group 3 used the cream        daily for four weeks after which they were asked to return the        cream and continue their normal regime observing the exclusion        criteria. Subject evaluations were conducted at initial visit,        weeks 2 & 4, and again at weeks 6 and 8 to facilitate capture of        “rebound” data four weeks after product application had ceased.

Daily application of the BiovaDerm™ has demonstrated:

-   -   28% reduction of deep wrinkles within 4 weeks in Group 3.    -   19% reduction of deep wrinkles within 2 weeks in Group 2.    -   30% reduction of deep wrinkles within 1 week in Group 1.    -   Effectiveness of treatment continued beyond two weeks from last        product application.

The positive effect was long-lasting and continued after use of thecream was discontinued. 1-2 weeks after cessation of treatment the skinstarted to revert to its pre-treatment state. It was observed thatmeasurable effect of the cream was still discernable after 4 weeks inwrinkle reduction, improved skin smoothness and luminance and reducedacne. Subject comments corroborated these clinical observations indetail. There were no negative/adverse observations from study subjectsor clinical staff.

Treatments & Mode of Administration Topical-BiOvaDerm™: Hydrolyzed eggmembrane powder of Bio2 in a cream base was applied to the face ofSubjects who have aged and/or sun damaged skin. After skin has beencleaned and dried in the morning and evening (at least 8 hours betweenapplications), subject removed 1FTU (finger tip unit) from one ouncejar, and applied dab of cream to face, covering temple to outward cornerof eyes, and around lips and chin where fine lines are found.

Study Endpoints

The effectiveness endpoint was the week 2, 4, 6, and 8 measurements offacial skin wrinkles. The principal investigators utilized aMoritex/BTBP skin analyzer which scans the face and sends quantitativedata to specialized software which calculates the number of, and extentof wrinkle severity and depth, Skin Complexion Health, Skin Texture andlaxity/tone changes, and UV damage.

Effectiveness Analysis

The effectiveness of the treatment was assessed by measuring the changein the prevalence and depth of wrinkles, the complexion health; rednessof the skin, and the smoothness and tone of the face before, during, andafter use of BiOvaDerm™ (Bio2) Cream.

Subjects

Subjects 18 years of age or older who had aged and or sun damaged skinwere qualified for participation in the trial, according to specificinclusion and exclusion criteria. Subjects who met all of theeligibility criteria were invited to participate. Study personnel(clinical investigator or research nurse/coordinator) explained thestudy to each subject, including procedures involved in the treatment,risk and benefits, alternative treatments and his/her rights as a studysubject. The subject was given the consent form and allowed as much timeas he or she required to read and decide on participation. Subject whodecided to participate, were asked to sign the consent form. Subjectswho signed the consent form and received study treatment were consideredenrolled. Subjects who withdraw after signing the consent but before anystudy treatment were recorded on a dropout log.

Subjects Inclusion Criteria

1. Subject must be 18 years of age.

2. Subject must be available for and willing to attend all evaluationvisits.

3. Subject must be able and willing to give informed consent.

4. Subjects should have aged and/or sun damaged skin, determined bysubjective observation.

5. Subjects must be willing to use appropriate birth control forduration of trial.

Subjects Exclusion Criteria

-   -   1. Subjects should not be utilizing accutane, or other skin        treatment drugs or topicals such as Isotretinoin, oral        antibiotics, topical antimicrobials, (Azelaic acid, Benzoyl        Peroxide, Clindamycin, Erythromycin, Sodium Sulfacetamide, or        topical retinoids, like Adapalene, Tazarotene, or Tretinoin)    -   2. Subject has known allergy to eggs or egg products.    -   3. Pregnant and breastfeeding women.    -   4. Subject is unwilling to forgo dermatologic or Aesthetic skin        treatments for the duration of the study.    -   5. Subject is involved in any other research study involving an        investigational product (drug, device or biologic) or a new        application of an approved product, within 30 days of screening.

Criteria for Evaluation

Subjects were given the face cream and instructed to apply it twice perday as a thin layer on the face in the morning and before bed at night.At two week intervals, the subjects attended the clinic at which timethey were asked to wash their faces to remove any makeup, oil or creamand their faces were scanned with the Moritex/BTBP skin analyzer(Clarity Pro Face Scanning System) and data pertaining to skin andwrinkles was obtained. The analyzer measures facial wrinkles andquantifies deep wrinkles, fine wrinkles and emerging wrinkles inseparate categories by computerized image analysis of wrinkle lines invery high resolution photographs, and skin color, smoothness tone,excessive oil, and UV damage of the face. The data was gathered byclinicians trained on the use of the face scanner. With few exceptionsall data was gathered by the same clinician in the same clinicenvironment.

Summary Findings

Application of the BiOvaDerm™ (BIO2) cream resulted in a quite positiveand sometimes dramatic reduction in wrinkles, and other skin conditions.(See charts below) The positive effect was long-lasting and continuedeven after use of the cream was discontinued.

After 1-2 weeks the skin started to revert to its pre-treatment state,but even after 4 weeks the effect of the cream was still discernible asa reduction in wrinkles, an improvement of skin smoothness and luminanceand reduced acne. Subject comments corroborated these observations indetail.

There were no negative/adverse observations from Clinical staff, orcomments from study subjects.

Adverse Effects & Safety Monitoring

Subjects were also instructed to record any changes in overall health,as well as any discomfort associated with the application of the cream.Subjects were asked to bring their diary to all evaluation visits forreview with the investigators. The subject self-assessment diary wasexamined at each visit providing the opportunity to note and discusswith the subjects any discomfort or other adverse events that might havebeen recorded. In addition, at baseline, week 2, and week 6, bloodchemistry, CBC, and other lab results were taken. Heart rate and bloodpressure measurements were also taken and results were recorded. PI orClinic staff noted any event or result which fell outside normal range,or expectation, (related or not related to study) the PI or Clinic staffwere instructed that should an event be observed, they were to providesuch information to the subject and recommended that they discuss thefindings with their private physician.

Effectiveness Analysis

Topical cream Effectiveness scan evaluations occurred at week 2, week 4,and week 6 following initiation of treatment in each subject. At theevaluation visit the investigator conducted face scans and computerizedanalyses of aged and/or sun damaged skin to assess improvements incomplexion health, pore & pigment health, redness, or areas where therewas UV damage.

The clinician used a Moritex/BTBP Skin Analyzer (Moritex/BTBP, Inc., SanJose, Calif.) which scans the face using high resolution photography andderives highly quantitative data pertaining to above describedconditions. By making one or two baseline scans per subject, it waspossible to quantify future changes in facial skin for subjects applyingthe cream in the study.

Statistical Considerations

Subjects were not randomized. Subjects were recruited to participate whohad aged and sun damaged skin, and who agreed to utilize the BiOvaDerm™(Bio2) Topical skin treatment formulation. A smaller number of normalyounger subjects were also recruited.

Results

A total of 23 subjects were recruited and enrolled in the trial.Clinician used a Skin Analyzer (Moritex/BTBP) which scans the face andsends quantitative data to specialized software which uses advancedimage analysis techniques to quantify the status of facial skin.

The study population varied in age and amount of wrinkles. Actualwrinkle index values were of limited use for comparative purposes and sofor presentation all data are presented as percentages of the baselinemeasurements for the population at the start of the study

Assessment of Skin Health Results Effect of BiOvaDerm™ Cream on Wrinklesand Facial Redness

TABLE 1 Effect of BiOvaDerm ™ Cream use on Wrinkles and Redness 1 2 3 45 Baseline Week weeks weeks weeks weeks Face Emerging(E) 100% 81% 81%95% 94% 99% Cream 1 Fine (F) 100% 83% 92% 83% 108%  87% week use Deep(D) 100% 70% 95% 82% 80% 48% Redness 100% 110%  102%  110%  87% 104% (E + F + D) 100% 76% 90% 86% 91% 73% n = 6 4 2 4 1 3 1 2 4 6 8 BaselineWeek weeks weeks weeks weeks Face Emerging(E) 100% 83% 85% 101%  Cream 2Fine (F) 100% 89% 84% 98% weeks use Deep (D) 100% 81% 83% 100%  Redness100% 99% 105%  100%  (E + F + D) 100% 84% 84% 100%  n = 9 9 7 4 FaceCream 4 Emerging(E) 100% 78% 101%  81% 98% weeks use Fine (F) 100% 110% 107%  106%  107%  Deep (D) 100% 93% 72% 76% 87% Redness 100% 112%  106% 114%  113%  (E + F + D) 100% 92% 87% 83% 94% n = 8 8 8 8 8

-   -   FIG. 11 is a graph depicting the average percentage change from        baseline wrinkle measurements for 8 subjects who used the cream        for 4 weeks and were followed an additional four weeks after        stopping use.

A general overview of the effects of the treatment can be seen in thegraph depicting the average percentage change from baseline wrinklemeasurements for 8 subjects who used the cream for 4 weeks (FIG. 11).Subjects discontinued use of creams at week 4 as is indicated by thevertical line. The data for weeks 6 and 8 represent values measuredafter use of product had been discontinued.

This data, representing group 3, demonstrated that use of the creamresulted in:

-   -   28% reduction of deep wrinkles after 4 weeks of daily use    -   17% overall reduction in all wrinkles (together) at 6 weeks (two        weeks after cessation of use).

As was anticipated, the use of the cream resulted in an increase of finelines concordant with the observation that Emerging, Fine and Deepwrinkles are not independent since loss of deep wrinkles can increasefine lines, and similarly, loss of fine lines can increase the measuresof emergent lines. Due to the nature of the face scanning device,measurement error was also expected to be greatest in emergent wrinklemeasurements followed by Fine lines and then Deep wrinkles.

-   -   FIG. 12 is a Graph depicting the average percentage change from        baseline wrinkle measurements for 9 subjects over who used the        cream for 2 weeks and were followed for another four weeks after        stopping use.

Group 2 subjects discontinued cream application after two weeks. These 9subjects showed similar changes (10%) to those in Group 3 (8%) for AllWrinkles [E+F+D] after two weeks of application. Subjects were asked todiscontinue use of creams after 2 weeks and this is indicated by thevertical line. The data for weeks 4 and 6 represent values measuredafter use of product had been discontinued.

This data, representing Group 2, demonstrated:

-   -   19% reduction of deep wrinkles after 2 weeks of daily use    -   15% overall reduction in all wrinkles two weeks after cessation        of use.

As was anticipated, the use of the cream resulted in an increase of finelines concordant with the observation that Emerging, Fine and Deepwrinkles are not independent since loss of deep wrinkles can increasefine lines, and similarly, loss of fine lines can increase the measuresof emergent lines. Due to the nature of the face scanning device,measurement error was also expected to be greatest in emergent wrinklemeasurements followed by Fine lines and then Deep wrinkles.

-   -   FIG. 13 Graph depicting the average percentage change from        baseline wrinkle measurements for 6 subjects who used the cream        for 1 weeks and were followed for another four weeks after        stopping use.

Group 1 subjects discontinued cream use after 1 week. These 6 subjectsshowed a reduction in all categories of wrinkles (24% All Wrinkles[E+F+D]). Subjects discontinued use of creams at week 1 and this isindicated by the vertical line. The data for weeks 2, 3, and 4 representvalues measured after use of product had been discontinued.

Complexion

The Tables below summarize the face scan analyses as percentage changefrom Baseline scans done at commencement of the study. Three variablesrelevant to Complexion are Pigment Evenness, Redness and Luminescence.Each table summarizes the results for each Group of subjects who usedthe cream for different lengths of time. All subjects were followed for4 weeks after stopping daily use of the cream.

1 2 3 4 Group 1 Baseline Week weeks weeks weeks 5 weeks Face CreamPigment Evenness 100% 101% 128% 117% 102% 148% 1 week use Redness 100%127% 118% 129% 101% 121% Luminescence 100%  92%  84%  90%  67%  99% n =6 4 2 4 1 3

1 2 8 Group 2 Baseline Week weeks 4 weeks 6 weeks weeks Face Cream 100%108%  95% 247% Pigment Evenness — 2 weeks use 100% 113% 123% 149%Redness — 100% 109% 113% 229% Luminescence — 9 9 7 4 n =

1 2 4 6 8 Group 2 Baseline Week weeks weeks weeks weeks Face Cream 4Pigment 100% 109% 125% 114% 117% weeks use Evenness Redness 100% 112%110% 114% 113% Luminescence 100% 103% 103% 100%  99% n = 8 8 8 8 8

Excess Sebum, Acne, and Pore Inflammation

The Tables below summarize the face scan analyses as percentage changefrom Baseline scans done at commencement of the study. Three variablesrelevant to Pore Health are Sebum level (oiliness), Acne and PoreInflammation. Each table summarizes the results from Groups of subjectsthat used the cream for different lengths of time. All subjects werefollowed for 4 weeks after stopping daily use of the cream.

1 2 3 4 5 Group 1 Baseline Week weeks weeks weeks weeks Face Cream 1Excess Sebum 100%  110%  123%  204%  110%  57% week use Acne 100% 48%48% 60% 82% 37% Inflammation 100% 57% 67% 43% 63% 13% n = 6 4 2 4 1 3

1 2 4 6 8 Group 2 Baseline Week weeks weeks weeks weeks Face Cream 2Excess Sebum 100%  79% 80% 10% weeks use Acne 100% 123% 56% 28%Inflammation 100% 181% 92% 92% n = 9 9 7 4

1 2 4 6 8 Group 3 Baseline Week weeks weeks weeks weeks Face Cream 4Excess Sebum 100% 110% 112% 68% 79% weeks use Acne 100% 115% 102% 95%78% Inflammation 100% 134% 148% 115%  138%  n = 8 8 8 8 8

Redness increased in all groups and did not appear to be affected bycessation of cream use. Luminescence and Pigment Evenness alsoincreased, resulting in a discernible smoothness, color and patina tothe skin in some subjects.

Use of BiOvaDerm™ Cream appeared to have a direct effect on Pore healthrelated parameters but less pronounced effect on Complexion. Changes inPore Health parameters appeared to be more closely correlated with theuse of the cream. Pore inflammation and acne as measured by the scanner,both increased during the period of cream use and decreased when creamapplications were discontinued falling to levels below the initial valuefor these measurements. The net effect therefore was an improvement inPore health and reduction in excess sebum (blocked pores), Acne andrelated pore inflammation.

-   -   FIGS. 14-15: Pore Health and Complexion related measurements.        [Read from “0” baseline to the vertical line which represents        the stopping point]

Improvements in redness, pigment evenness, and luminescence allincreased over the course of the study and were commented on by subjectsin interviews and diaries. The effect on these parameters seemed tocontinue forward as even after subjects stopped applying the cream, anupward trend in skin complexion and health was observed.

Discussion Wrinkle Count, Prevalence and Severity:

Subjects in the study ranged in age from their twenties to sixties.Subject by subject, the actual indices for the three sub-categories ofwrinkles varied widely. To compare and contrast the measurements, thedata was expressed as percentages of the baseline measurement for eachsubject. The Baseline measurement (made at commencement of the study)was set at 100% and all subsequent measurements compared to thatstarting value. Summary tables and graphs included present the groupaverages of those changes.

Wrinkles decreased in the majority of subjects in response to creamapplication within one week. The response times varied among thesubjects and some subjects showed no apparent wrinkle reduction. Aninteresting finding is that the data suggests a linear relationshipbetween amount of time treatment is applied and biological effect overthis time frame. The average reduction of wrinkles after 2 weeks of usewas approximately 50% of the reduction seen after 4 weeks.

Changes in wrinkle indices are not independent since they mayinterconvert in response to treatment. In wrinkle improvement, Deepwrinkles are expected to smooth out into Fine wrinkles, which convert toEmergent wrinkles (and vice versa when cream application is stopped).This suggests an explanation for the two week lag between the maximumeffect of Deep wrinkles versus the maximum effect of combined (AllWrinkles summed) measurements.

The results obtained in this study are highly significant fromscientific, cosmetic, and marketing perspectives when compared to otherpublished studies of anti-wrinkle creams. A very highly publicizeddouble blind randomized controlled study carried out in the UK (Watsonet al., 2009) in which subjects with aged skin used a plant basedanti-aging formulation, it was found that at 12 months, 70% of theparticipants showed an improvement in facial wrinkles compared to thestart of the study versus a statistically modeled null prediction of 33%of people expected to show improvement using the control (vehicle) creamfor 12 months. The improvement in the test group was described as“clinically significant” but researchers did not observe any otherbenefits of the test product over the control. For example, there wereno improvements in the amount of abnormal coloration of the skin andboth the test and control products produced similar improvements in skinroughness from the start of the study (see this article from theNational Health Services Choices;www.nhs.uk/news/2009/04April/Pages/Facecreamreduceswrinkles.aspx, of theUnited Kingdom).

-   -   FIG. 16: Sum of wrinkle measurements (Deep+Fine+Emergent) for        six subjects expressed as percentage of initial pre-use value.        Cream use was started day 1 and discontinued after 2 weeks        Group 2. An overview of the effects of the treatment can be seen        in the graph of Total Wrinkles index (Deep+Fine+Emergent) over        the course of the study. The wrinkle data are plotted as        percentages of the value calculated for the baseline scan taken        during first visit 1 for this group (i.e. before the subjects        started the treatment). Subjects were asked to discontinue use        of creams at 2 weeks to test effect of non-use after daily        application. The data for later visits represent wrinkles        analysis after product use had been discontinued. In this group,        use of the cream resulted in a reduction of wrinkles that was        detected after 2 weeks of daily use and varied from 5% to 63%        reduction among the subjects. KEY: JR, LE, NR, RC=Initials of        study subjects

Effect of BiOva Cream on Deep Wrinkles

-   -   FIG. 17:    -   Example of wrinkle measurements (Deep). Four subjects expressed        as percentage of initial pre-use value (day 1). Cream use        started day 1 and discontinued at 2 weeks. In these subjects,        use of the cream resulted in reduction of deep wrinkles detected        after 2 weeks of daily use and varied 20% to 60% reduction among        the subjects. When cream was discontinued, (at 2 weeks), the        skin started to revert to its pre-treatment state was seen over        the following 4 weeks.

Effect of BiOva Cream on Fine Lines

-   -   FIG. 18: Wrinkle measurements (Fine) for four subjects expressed        as percentage of initial pre-use value. Cream use was started on        day 1 and discontinued after 2 weeks.

In most subjects, use of the cream resulted in a reduction of wrinklesthat was detected after 2 weeks of daily use and varied among thesubjects. When use of the cream was discontinued, (Visit 3), the skinstarted to revert to its pre-treatment state over the following 4 weeks(visits 4 and 5). When analyzing deep wrinkle reductions, the scannertends to identify more of the fine wrinkles, as a result the effect isless clear in this measurement category. After discontinuing cream usefor a month, only 7 subjects in the study had returned to valuesstatistically the same as their starting values (i.e. >90% of theirbaseline measurement). All other study subjects had wrinkle valuesranging from 44%-90% of their baseline measurement.

Effect of BiOva Cream on Emerging Wrinkles

In most subjects, use of the cream resulted in a reduction of wrinklesthat was detected after 2 weeks of daily use and ranged from 5% to 50%reductions among the subjects. When use of the cream was discontinued,(Visit 3), the skin started to revert to its pre-treatment state overthe following 4 weeks (visits 4 and 5). After discontinuing cream usefor a month, only 7 subjects in the study had returned to valuesstatistically the same as their starting values (i.e. >90% of theirbaseline measurement). All other study subjects had wrinkle valuesranging from 44%-90% of their baseline measurement.

Complexion Health

Complexion health showed specific trends in response to cream use:

-   -   Redness increased in all three groups and stayed elevated        throughout the study. It did not appear to be directly affected        by cessation of cream use, suggesting an increase in        perfusion/skin health/circulation.    -   Evenness of pigment increased significantly.    -   Luminescence of the skin, another measure of smoothness, showed        less treatment related variation.    -   FIG. 12: Wrinkle measurements (Emerging) for four subjects        expressed as percentage of initial pre-use value. Cream use was        started day 1 and discontinued after 2 weeks.

Skin Texture and Laxity Changes

The values for emergent wrinkles are representative of skin texture.While emergent wrinkles clearly showed a response to the treatment itwas more variable among subjects. As emergent wrinkles are close to thenormal texture of skin, they are more prone to confounding factors likeskin hydration and measurement error from positioning of the subject inthe scanner, etc. The machine used for measurements in this study wascalibrated to accept variations in facial position potentiallycorresponding to +/−5%-8% variation in the wrinkle index [Moritex/BTBPScanner manufacturer's personal communication].

Pore Health

Use of BiOvaDerm™ Cream appeared to have a direct effect on Pore healthparameters but less pronounced effect on complexion. Redness appeared toincrease slightly in all groups and did not appear to be affected bycessation of use, the effect likely due to increased microcirculation,(increased circulation to skin surfaces) enabling an exchange of water,oxygen, carbon dioxide, and nutrients, between blood an surroundingtissues, an important component of skin health, increasing luminescenceand smoothness.

Changes in Pore Health parameters appeared to be more closely correlatedto the use of the cream. Pore inflammation and acne as measured by thediagnostic scanner documented that both decreased during the period ofcream use and changed the amount of sebum and affected the smoothing andoverall clearing of the skin.

UV Damage

Subjects in the study had sun damage ranging from moderate to severe. Inthe majority of subjects this remained approximately constant throughoutthe study and did not appear to be directly affected by the use ofBiOvaDerm™ Cream.

APPENDIX—A

Tables of averaged indices data for different face analysismeasurements. Each table contains the data from one of the three groups(Group using the cream for 4 weeks, 2 weeks and one week respectively).

Four Week Use Group

Averages 0 1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8weeks emerging 44.25 34.38 44.75 35.88 43.5 fine 30.13 33.25 32.25 3232.13 deep 79.88 74.38 57.38 60.5 69.5 redness 23.18 25.9 24.51 26.3426.16 All Wrinkles 153.75 142 134.38 128.38 145.13 Pigment evenness13.66 14.9 17.01 15.63 16.01 Redness 23.18 25.9 25.45 26.38 26.09Luminescence 25.14 25.81 25.81 25.24 24.79 Excessive Sebum 602.75 661.88677 407.25 477.75 P. acnes 1143 1312.81 1165.63 1082.5 889.88inflammation 416.63 558.13 615 477.13 576.75 Deep Inflammation n = 7 8 88 8 8 8 8 8

Two Week Use Group

Averaged values for 0 1 2 3 4 5 6 emerging 57.89 44.6 48.22 49.43 8053.33 fine 39.78 27.8 35.22 33.57 56 29.33 deep 56 34.7 41.9 49.43 57.538.33 redness 27.41 43 35.33 28.7 11.85 25.83 sum wrinkles 153.67 85.6113.78 132.43 193.5 121 Pigment evenness 14.13 20.94 15.27 13.37 16.334.9 Redness 23.86 27.12 26.9 29.36 11.85 35.5 Luminescence 24.23 44.4626.34 27.27 28.95 55.4 Excessive Sebum 521.86 402.25 412.63 417.17 1150.5 P. acnes 729.57 345.5 900.5 408.83 127 204.5 inflammation 286.07111.25 518.56 262 175 264 Deep Inflammation n = 1 9 5 9 0 7 2 3

One Week Use Group

Averaged values for 0 1 2 3 4 5 6 emerging 64.75 51.17 48.75 49 73 5759.67 fine 41.25 37.5 36.25 40 48.5 47 37.67 deep 83 67.5 60.25 81.5 9769 41 redness 22.1 23.93 24.73 22.85 27.9 19.5 23.37 sum wrinkles 189156.17 145.25 170.5 218.5 173 138.33 Pigment evenness 16.88 3.35 17.7322.45 25.18 17.8 25.9 Redness 11.78 0 11.6 16.6 20.08 0 16.77Luminescence 26.4 3.02 22.88 20.75 27.3 16.6 24.43 Excessive Sebum259.38 38.33 278 312.5 586.25 280 144.67 P. acnes 496.25 55 220 220351.25 380 171.67 inflammation 421.25 51.67 228.75 267.5 236.25 250 53Deep Inflammation 0 0 0 0 0 0 0 n = 4 6 4 2 4 1 3

APPENDIX—B

Face diagnostic-scanning equipment used in study.

Clarity™ Pro Face Scanning System (Moritex Incorporated, USA)Quantitative Analysis of Skin Condition. Clarity™ Pro features earlyskin condition detection, statistical comparison of before and aftertreatment images, and product and treatment recommendations. This systemis integrated with the Facial Stage, capturing digital, high resolutionfull face images for recognition and extraction of particular skincondition features. Images are taken in a dual light mode: true whitelight and safe UV capture.

REFERENCES

-   1. Baker, J. R. and Balch, D. A., Biochem. J., 82: 352-361, 1962-   2. Picard, J., Paul-Gardais, A., and Vedel, M, Biochimica et    Biophysica Acta, 320: 427-441-   3. Gautron, J., et. al, Connective Tissue Research, 42:255-267, 2001-   4. Starcher, B. C. and King, G. S., Connective Tissue Research,    8:53-55, 1980-   5. Akagawa, M, et. al, Biochim. Biophys. Acta, 14:151-160, 1999-   6. Hincke, M. T., et. al, Matrix Biology, 19:443-453, 2000-   7. Deal, C L and Moskowitz, R W, Osteoarthritis, 1999. 25:379-395-   8. Matheson, A J and Perry, C M, Drugs Aging. 2003; 20:1041-1060-   9. Kevin J Ruff, Anne Winkler, Robert W Jackson, Dale P Devore,    Barry W Ritz; Clinical rheumatology. 2009 Apr. 2-   10. Sim, J. S., S. Nakai, and W. Guenter, editors—Egg Nutrition and    Biotechnology, CABI Publishing, UK 2000 pp 219-232

Example 14 Human Clinical Safety and Efficacy Pilot Study for theTreatment of Knee Associated Musculoskeletal Pain and Stiffness

309 was evaluated for safety and efficacy in the treatment ofmusculoskeletal pain and flexibility. For study purposes, the knee jointwas specified and monitored for the 6 week duration of the study.

An open-label Pilot Study was conducted. Forty-two subjects wererecruited and assigned to receive 450 mg of BiovaFlex™(BIO2) daily for 6weeks. Subjects were clinically evaluated at established intervals forboth effectiveness (pain level and total flexion) and overall healthevaluations. Assessments were made at 2, 4 and 6 weeks. Daily onlinetracking and measurement of the subject's joint pain was recorded viathe Western Ontario and McMasters University (WOMAC) Abbreviated Index.

-   -   Pain symptoms were reduced 8.25% from base line within 7 days        and 16.42% from baseline within 14 days.    -   Total pain symptoms were reduced 20% from baseline at study end.        The average WOMAC value across all subjects fell from 4.61        (baseline) to 3.69 (study end). A reduction of 0.97 or 20.78%.        (P<0.0001)    -   This reduction in absolute pain is significant in that the pain        relief overcame the pain increases normally associated with        increases in flexion. Improved Knee Functionality in just seven        days was 9.191%, and 10.14% from baseline within 14 days.    -   Relative Knee Functionality improved 37.8% over the course of        six weeks.    -   The safety profile was remarkable with no reported adverse        events.

This study shows that BiovaFlex™(BIO2) is an effective and safealternative treatment for musculoskeletal pain and stiffness asassociated with the knee.

This study was designed to assess the effectiveness of theBiovaFlex™(BIO2) compositions in the form of an ingestible dailysupplement for the treatment of connective tissue and joint pain anddiscomfort.

Biova LLC in Johnston, Iowa. supplied Clinic Testing site with capsulescontaining BiovaFlex™(BIO2) in the following: 220 small bottles with 40capsules, each containing 150 mg of 309. The studied dosage required theconsumption of 3 capsules daily to total 450 mg per day. Approximately atwo week supply of product per bottle and a 6 week supply of capsules intotal were provided to each subject over the duration of the study.

Product Accountability

Supplies delivered to the investigational Clinic were distributed toenrolled subjects at each visit. All supplies were labeled by the Clinicwith study date, Clinic name, Supplement name, a number from 1 to 45,with use/dose instructions. Later when the product was supplied to thesubject, the subject's identifier code was written on the bottle/jar.The site used a form to document distribution of capsule supplies amongsubjects. This form was maintained by the investigator. If a subjectwithdrew from the study, any remaining capsules and capsule containerswere collected by the investigator. An accurate account of capsulesconsumed by each subject in the study was maintained.

Intended Use

The Ingestible Natural Eggshell Membrane supplement of BiovaFlex™(BIO2)is intended for treatment of the discomfort from connective tissue andknee joint pain. Hydrolyzed eggshell membrane powder of BiovaFlex™(BIO2)for the study was contained in capsules of 150 mg per capsule. Three (3)capsules are taken by mouth along with water or juice daily.

Study Design

The study was conducted to evaluate the safety and efficacy ofBiovaFlex™(BIO2) ingredient on joint and musculoskeletal pain. Forty twosubjects were enrolled in a period of approximately one month andevaluated over the course of 6 subsequent weeks at the test center inSausalito, Calif. (USA). Selection was based on health and physicalevaluations of the subjects along with satisfaction of theinclusion/exclusion criterion set forth in the design of the study.Provision of written informed consent was mandated and obtained prior toenrollment of the subjects in the study. Treatment involved a 450 mgdaily dose of BiovaFlex™(BIO2) soluble eggshell membrane powder viathree 150 mg capsules.

Clinic visits and assessments were scheduled at weeks 2, 4 and 6 fromsubject start. Subject compliance reviews were performed by subjectinterviews and via monitoring of the subject's online daily diary andWOMAC surveys. Subjects were asked to log questions and or concerns ifnecessary, in dairies provided to them.

Subjects 18 years of age or older with history of arthritic type jointpain or connective tissue disorder with a reduced range of motion wereconsidered for this study (1). In the selection process the potentialsubjects clinical history was reviewed and vital signs observed.Selection was based on subject fulfilling the inclusion criteria anddisqualifying others based the exclusion criterion. Subjects wereincluded based on Inclusion Criteria. (see below) Subjects acknowledgedpersistent pain in one or both knees or complained of connective tissuedisorder with an assessment score of at least 5, but not more than 9using the Pain Assessment and Pain Intensity Rating scale. Subjectsagreed to replace current pain relief medications with BiovaFlex™(BIO2)treatment, and female participants agreed to use appropriate birthcontrol for duration of trial.

Subjects were excluded (see detail below) if they had a history ofRheumatoid Arthritis, or were on medications such as methotrexate orimmunosuppressive medications. Subjects were asked to refrain fromtaking any other vitamins or supplements.

Subjects who met all of the eligibility criteria were enrolled toparticipate. As part of their informed consent subjects were informed oftheir rights as research participants; the contents of the study,including the procedures involved and the treatment, risk and benefitsassociated with it, along with any other alternatives available wereexplained to them. A signed consent form was required and received inorder to allow the subjects to participate. A “drop out” log wasmaintained for any participant who dropped during the duration of thestudy.

Treatments & Mode of Administration

Subjects were instructed to take a daily dose of three 150 mg (450 mg)processed BiovaFlex™(BIO2) with juice or water as treatment for theirmusculoskeletal pain and stiffness. Subjects were evaluated on-site atWeek 2, Week 4, and Week 6, and their supplement was replenished tillthe duration of the next visit. Compliance was tracked using multiplemethods including: subject's daily self-assessment diary, to be filledthroughout the study period; an online self-assessment electronicquestionnaire for pain and stiffness assessed at various range ofmotions, the WOMAC Assessment and Pain Intensity Rating Scale; or theCenter's Study Compliance Phone Line, where subjects identify themselvesand record comments regarding their condition. Clinic staff alsofollowed-up with subjects by telephone.

The primary endpoint was to evaluate the effectiveness of 450 mgBiovaFlex™(BIO2) as a treatment for pain, stiffness, and discomfort forknee associated musculoskeletal disorder. Subjects were evaluated forresponse to treatment in clinic on Week 2, Week 4, and Week 6. Duringeach clinic visit, review of medical history, physical evaluation, andpatient interview provided insight to patient compliance and concernswith respect to the treatment. In addition, subjects were interviewedabout situations that have potential to mask symptoms or produce adversereactions, such as recreational drug abuse or applied-therapeutics orsupplements. Effectiveness of response, via knee exam, was determinedthrough pain assessment at rest and on motion as measured on a numericalscale ranging from 0 (no pain) to 10 (worst pain perceivable), alongwith range of motion as measured using a Goniometer. Signs and Symptoms,e.g. swelling, crepitus, were documented to provide insight into thefunctionality of knee. The online WOMAC Pain Assessment and PainIntensity Rating scale was also used to quantify the pain and stiffnessand provided a objective tool to evaluate subjects in-between visits.Primary endpoint values were compared to baseline (first visit) todetermine efficacy of treatment.

Adverse Effects & Safety Monitoring

The secondary endpoint was to assess the safety of BiovaFlex™(BIO2)capsules as a treatment option for knee associated musculoskeletaldysfunction. Subjects were also instructed to record any changes inoverall health, as well as any discomfort associated with ingestion ofthe capsules in their self-assessment diaries. An evaluation of heartrate and blood pressure was performed with every visit, to keep track ofany potential adverse cardiovascular effect. The subject self-assessmentdiary was reviewed at each visit and any discomfort of other adverseevents recorded was discussed with each subject. Blood Chemistry,hematology, and allergen antibody assays provided valuable insights inany systemic changes that might be of negative consequence to thesubject. The testing was performed by a CLIA certified, medicaldiagnostic laboratory and included non fasting glucose, sodium,potassium, calcium, chloride, carbon dioxide, urea, creatine, alkalinephosphatase, total protein, albumin, globulin, AST, ALT, and totalbilirubin. Blood samples from subjects were drawn by certifiedphlebotomists on initial visit, at week 6 and at varying intervals inbetween.

Inclusion Criteria

Subject selected were 18 years of age or older; able and willing to giveinformed consent and attend all evaluation visits. Subject hadpersistent pain in one or more knee joints or connective tissue with apain assessment score of at least 5, but not more than 9 using the PainAssessment and Pain Intensity Rating scale. Subject agreed to take/useBiovaFlex™(BIO2) compositions to replace current pain reliefmedications, and was willing to use appropriate birth control forduration of trial if necessary.

Exclusion Criteria

Subjects with Rheumatoid Arthritis, or currently receivingremission-inducing drugs such as methotrexate or immunosuppressivemedications in the past 3 months, or using accutane or other skintreatment products were excluded. Subjects using accutane, or other skintreatment drugs or topicals such as Isotretinoin, oral antibiotics,topical antimicrobials, (Azelaic acid, Benzoyl Peroxide, Clindamycin,Erythromycin, Sodium Sulfacetamide, or topical retinoids, likeAdapalene, Tazarotene, or Tretinoin were also excluded. Subjects with apersistent pain score >9 using the Pain Assessment and Pain IntensityRating scale were excluded from the joint study arm but were allowed toparticipate in the Skin Treatment arm of the study.

In addition, subjects were excluded if they had known allergy to eggs oregg products; were Pregnant or breastfeeding women; unwilling to forgouse of over-the-counter (OTC) treatments for the of the study; involvedin any other research study involving an investigational products (drug,device or biologic) or a new application of an approved product, within30 days of screening; were in poor health, diabetic, high bloodpressure, a heart condition, had known problems with bleeding, onmedications to stabilize organ function, or moods, or were over-weightby 20% to 25% their maximum desirable weight for their height. Subjectswere required to refrain from taking any other vitamins or supplements.

Subjects who met all of the eligibility criteria were invited toparticipate. Study personnel (clinical investigator or researchnurse/coordinator) explained the study to each subject, and reviewedstudy procedures involved in the treatment, risk and benefits,alternative treatments and his/her rights as a study subject. Thesubject was given the consent form and allowed as much time as he or sherequired to read and decide on participation. Subject who decided toparticipate, were asked to sign the consent form. Subjects who signedthe consent form, and provided a medical history and met the inclusioncriteria and agreed to the treatment were considered enrolled. Subjectswho withdraw after signing the consent but before any study treatmentwere administered were recorded on a dropout log.

After a Consent Form was signed by the subject, baseline information wascollected and the following procedures were conducted:

-   -   Review of Medical history. including past or present symptoms,        drugs-therapeutic and/or abuse, operations, trauma, joint pain        History and Physical exam-functional evaluation        (work/walking/swelling/how alleviated) including; knee        exam—noting swelling, walk with a limp, needs to use a cane        and/or wheelchair, etc.    -   Scoring of joint pain or connective tissue pain using the WOMAC        Pain Assessment and Pain Intensity Rating Scale. Determine range        of motion of effected joint(s), a pain reduction of 0.97 or        20.78% (P<0.0001)    -   Concomitant medications.    -   Blood Chemistry, CBC, blood pressure and heart rate.

Study Schedule:

-   -   Table One. S=Supplement T=Topical.

Results

A total of forty-two subjects were recruited and enrolled in the trialwith respect to the selection criteria discussed above.

The average drop in pain over the study period across all individualswas almost one full point on the WOMAC scale: 0.97 (4.61 (baseline) to3.69, a pain reduction of 20.78% (P<0.0001) (study end)). (Also seediary data for some of the more dramatic effects) A full/point decreasein absolute pain is substantial because it means that even as degree offlex increases, the pain relief is so substantial that it outweighs theupward pressure on pain due to increase in flex. The functionalitymeasurements show increases in overall functionality of the knees givenboth flex and pain.

Treatment with BiovaFlex™ demonstrated:

In First 10 days of Treatment Pain Is Reduced an Average of 23.77% inall Subjects:

Going on up or At night While Walking on flat down stairs - while inbed - While sitting or standing - Average for surface - pain pain painlaying - pain pain All 23.64% 24.69% 18.92% 20.00% 31.58% 23.77%

The subject self-report data show that over a ten day period pain wasreduced by 23.64% for walking on a flat surface, 24.69% for going up ordown stairs, 18.92% while in bed, 20% while sitting or laying, and31.58% while standing. The average reduction in pain for all subjectsover all measured activities was 23.77%. This implies, if the set ofsubjects is truly representative of the set of consumers of the product,that a reduction in pain of 18.92% to 31.58% with consistent consumptionof the intervention product is not inconsistent with the data. Thisanalysis is based on average responses from survey data reported bysubjects based on the WOMAC pain scale.

Toxicity and Adverse Effects

Medical Histories Review

Twelve subjects dropped/no showed from the study due to a combination ofpersonal reasons, exclusionary or precautionary measures taken on byclinical staff.

Dropouts:

CODE REASON 1 LR 40 Blood Pressure - slight- hyperactive 2 JL 30 BloodPressure - white coat 3 JH 48 Personal 4 PT 39 On medication for BloodPressure 5 AG 4 Personal 6 TFB 3 Egg Allergy 7 CM 51 Egg Allergy 8 AS 44Personal (drug usage) 9 DW 50 Due to no show 10 DJ 49 Personal (Surgery)11 SM 45 Due to no show 12 AL 1 Personal (family)

Blood Analysis Results Summary

As part of the design to ascertain safety and efficacy, an average oftwo blood samples were taken from each subject. Blood samples werecollected at baseline at the end of the study. In addition, sampling wasdone at varying intervals throughout the treatment period to monitorallergies or toxicity. As a part of the safety protocol, bloodchemistry, hematology, and allergen antibody assays were performed. Thetesting was performed by a CLIA certified, medical diagnostic referencelaboratory.

The blood chemistry analysis included non fasting glucose, sodium,potassium, chloride, carbon dioxide, urea nitrogen, creatinine, calcium,alkaline phosphatase, total protein, albumin, and globulin. In addition,AST, ALT and total bilirubin were tested to monitor liver function.

A complete blood cell count was performed to monitor general systemfunction. The hematology analysis included a hematocrit and hemoglobinlevel, red cell, white cell, and platelet counts, as well as cellmorphology.

A slight overall rise in blood chloride and urea nitrogen were noted,but were still within the reference range at the study completion.

Although there was an occasional nonspecific result that fell outsidethe reference range, all results were predominantly well within normalranges. In review of analytical data reported, there was no significantchange from baseline beyond the normal variations that are expected.There was no negative effect nor toxicity observed in blood chemistryanalysis. Hematocrit and hemoglobin concentration showed little changeand there was no abnormal blood cell morphology. Furthermore, none ofthe participants showed any significant increase in IgE antibodies toegg allergens.

Effectiveness Analysis

The following will be a discussion of the data obtained from the patientdiaries and the flexibility/pain measurement data. These data are usedto analyze the overall effectiveness of the BiovaFlex™ product onsubjects over time. All data were collected according to guidelinesdetailed by the WOMAC™ scale. (cite:http://www.womac.org/womac/womac_userguide.htm) Diary data comprises theself-report segment of the study, while the Pain/Flexibility datacomprise corroborating medical measurements.

Subject Self-Report Logs

In addition to the data collected from subject blood work, subjects wererequired to note protocol compliance in an online electronic journal ona daily basis. Data collected include time, date, patient information,and pill consumption behavior. In addition, several questions from theWOMAC scale were presented, including the level of pain (mild, moderate,severe, extreme) for the following activities: walking on a flatsurface, going up or down stairs, at night while in bed, while sitting,and while standing. Space was left for comments to allow comparison ofresponse with subject qualitative notes. Subject self-report logs servedas positive reinforcement for protocol compliance. Further, thetabulation of this data illustrates some of the evolution of subjectresponses over time in a more granular fashion. Summary tabulations ofresults are displayed in Table Two below.

TABLE TWO Subject self report (online diary) data. The data show thechanges from the inception period to the end period. Numbers denote thepercentage of total individuals listing the response (none, mild,moderate, severe, extreme) on the pain questionnaire. SubjectSelf-Report Log Data None Mod. Severe Extreme Date Range Factor (%) Mild(%) (%) (%) (%) April 5 46.39 30.93 20.62 0.00 0.00 4 54.64 34.02 9.280.00 0.00 3 77.32 21.65 0.00 0.00 0.00 2 39.18 38.14 20.62 0.00 0.00 134.02 47.42 16.49 0.00 0.00 January 5 40.61 29.95 26.90 1.02 0.00 445.18 36.04 13.20 4.06 0.51 3 55.84 24.87 15.74 3.05 0.00 2 27.92 28.4327.92 13.71 2.03 1 39.09 30.96 25.89 3.55 0.00 Key to factors: 1 =Walking on flat surface - pain; 2 = Going on up or down - pain; 3 = Atnight while in bed - pain; 4 = While sitting or laying - pain; 5 = Whilestanding - pain.

A simple analysis of diary data over time showed that a smallerproportion of subjects showed pain in each question as participation inthe study changed. The percentage of subjects suffering from no pain infactor five went from 40.61% to 46.39%, in six week, showing apotentially positive redistribution from higher pain groups to lowerpain groups. The percentage of subjects in the worst pain groupsdeclined to zero at the end of the study.

TABLE THREE Subject self-report (online diary) data. This chartdocuments the changes in the percentages of subjects reporting variouslevels of pain from the inception of the study in January to the end ofthe study in April. The higher categories show decline while the “None”category shows increase in all but one area. None Mild Mod. SevereExtreme Factor (%) (%) (%) (%) (%) 5 12.45 3.168445 −30.45 TR TR 4 17.31−5.93768 −42.24 TR TR 3 27.78 −14.873 TR TR TR 2 28.73 25.45884 −35.40TR TR 1 −14.90 34.71109 −57.00 TR TR

As the data in Table Three show, the “Severe” and “Extreme” categoriesare completely vacated by the end of the study. The “Moderate” paincategory has shown significant decline. Declines are seen in the mildcategory and the “None” category has seen increases for all exceptfactor one. The bulk of increase for factor 1 appears to have beendistributed with the “Mild” category. This evidence is consistent withthe hypothesis that the BiovaFlex™(BIO2) supplement has a positiveeffect on the reduction of knee pain.

Direct Flexibility and Pain Scale Measurements

Supplement effectiveness evaluations were carried out at week 2, week 4,and week 6 following initiation of treatment. During the follow upevaluation visits the clinical investigators reviewed the subject'sdiary and conducted a pain assessment using the pain assessment and painintensity rating scale. The investigators also conducted manual range ofmotion assessments on the subjects knee(s) Another pain assessment usingthe WOMAC pain assessment and pain intensity rating scale was also madeduring range of motion measurements.

The mean baseline pain assessment score was calculated for all subjects.The change in pain assessment score for each treatment was calculatedand then averaged across the subject population at week 2, 4, and 6 ofthe evaluation.

A challenge when measuring the effects of knee pain is that there isnatural confounding in the end measured results: the knee's range ofmotion, i.e. degrees of flexibility and subject' pain level at specificmeasurements within that range of motion. The data set is essentiallysubject to double variables. Therefore, a simple analysis of eithermeasurement may show a fallacious positive or negative relationshipbetween the measurement targets and the intervention material. In orderto compensate for these double variables, a couple of simple formulaehave been developed to enable comparisons of data averages.

The formulae are as follows:

$\begin{matrix}{{{{{Relative}\mspace{14mu} {Functionality}\mspace{14mu} \left( {{Pop}.\mspace{14mu} {Average}} \right)\text{:}\mspace{14mu} Y} =};}{{\frac{1}{n}{\sum\limits_{i}^{n}{\frac{\left( \frac{x_{1i}}{90} \right)}{\left( \frac{\left( {10 - x_{2i}} \right)}{(10)} \right)}x_{2i}}}} \neq 10}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

This calculates the relative increase range of motion of leg given pain,or “Relative Functionality” of the subjects knees. It is simply thecorrected ratio of range of leg motion compared to the inverse of thelevel of pain, to denote pain-related functionality. The end-result is acomparison of percentages put in the same numerical terms, whichdelineates the degree to which increases in total functionality can beattributed to increased range of leg motion.

$\begin{matrix}{{{Total}\mspace{14mu} {Functionality}\mspace{14mu} \left( {{Pop}.\mspace{14mu} {Average}} \right)\text{:}\mspace{14mu} Y} = {{\frac{1}{n}{\sum\limits_{i}^{n}\left( \frac{x_{1i}}{90} \right)}} + \left\lbrack \frac{\left( {10 - x_{2i}} \right)}{\left. 10 \right)} \right\rbrack}} & {{Eq}.\mspace{14mu} 2}\end{matrix}$

This calculates the total increase range of motion of leg given painlevel, or “Total Functionality” of the subjects knees. It is thecorrected ratio of range of leg motion added to the inverse of the levelof pain, to denote pain-related functionality. The result is a correctedversion of the total change in subject knee functionality over the studyperiod.

In both equations please note the following: y denotes the estimatedoutcome measure, n is the number of subjects, i is the ith subject,x_(1i) is the degree of movement for subject i, x^(2i) is themeasurement on the pain scale for subject i, 90 is the total number ofdegrees of movement for a totally functional knee and 10 is the totalfor the pain scale. As 10 is the highest level of pain, 10—x_(2i)denotes the pain-related functionality of the subject.

Baseline Comparison Total Change Relative Functionality 1.5096992.080397 37.8% Total Functionality 1.135095 1.351762 19.1%

Source: This chart denotes population averages tabulated as per Eq. 1,Eq2.

The data obtained are supplied in the chart above. The data illustratenoticeable improvements over the three sampling time points, week 2, 4 &6. The interpretation of the information in the chart above is asfollows: relative flexibility fluctuated in the sample by 37.8%. Thismeans that of the amount of functionality gained, 37.8% as calculated,can be attributed to the relative increases in degree of knee bendconcurrent to the relative reductions in pain as realized within thisrange of motion. (Also, it is worth noting that the p-values for all ofthe values are p<0.0001, allowing for the rejection of the nullhypothesis.) This is consistent with the observation that as theintervention takes effect, the subject is able to increase his/her angleof motion to the point of pain tolerance. The increase in totalfunctionality means that knee functionality taking into account bothangle of motion and pain level have increased by a total of 19.1%. Thesecalculations are based on averages documented above computed on thesample of individuals provided in the study.

DISCUSSION-CONCLUSION

Chronic debilitating knee pain and dysfunction is an extremely commondisorder and is a significant cause of dysfunction in individuals of allage groups. This study was designed to evaluate the safety and efficacyof BiovaFlex™ as a treatment option for subjects suffering from kneeassociated musculoskeletal dysfunction.

The study concludes that BiovaFlex™ is a viable option for treatment ofknee and musculoskeletal disorder in that it has proven effectiveness injoint related symptoms and has been shown to be safe, with no reportedadverse effects. The absence of reported adverse events with BiovaFlex™may prove it to be more advantageous as a remedy than the traditionalroute of joint relief with NSAIDS, with their serious side effects.

Subjects have shown significant improvement in symptoms relative to theshort duration of the study. (an average drop in pain over the studyperiod across all individuals was almost one full point on the WOMACscale: 0.97, in just the first 10 days of treatment pain was reduced anaverage of 23.77% in all subjects.

As the data in Table 3 shows, in this 45 day study, there is asignificant decline in pain of moderate level with redistribution intolower pain groups. Relative functionality of 37.8% denotes a markedimprovement in overall flexibility. This shows that range of motion hasimproved with subjects showing greater degrees of flexion. Totalfunctionality of 19.1%, although not as marked as relativefunctionality, is nevertheless significant given the short duration ofthe study. This total functionality denotes improvement in symptoms onjoint usage (e.g. walking, climbing stairs). It is evident from theseresults that use of BiovaFlex™ does have potential as a treatment forsymptomatic joint relief in knee musculoskeletal dysfunction, more sowith its absence of reported adverse effect. This is significantespecially when dealing with a condition that requires long-termtreatment.

The improvements in the signs and symptoms may in part be related to theGlycosaminoglycans (GAGs), which are constituents of the BiovaFlex™.Glycosaminoglycans (GAGs), the class of amino sugars to which Hyaluronicacid, chondroitin-sulfate and glucosamine belong to, are an integralpart of the amorphic ground substance that constitutes the extracellularmatrix of connective tissues, for example articular cartilage. GAGS havehighly charged side groups that render them extremely hydrophilic, thusattracting large volume of water, which imparts the characteristicturgor of supportive tissues.

REFERENCES

-   1. Baker, J. R. and Balch, D. A., Biochem. J., 82: 352-361, 1962-   2. Picard, J., Paul-Gardais, A., and Vedel, M, Biochimica et    Biophysica Acta, 320: 427-441-   3. Gautron, J., et. al, Connective Tissue Research, 42:255-267, 2001-   4. Starcher, B. C. and King, G. S., Connective Tissue Research,    8:53-55, 1980-   5. Akagawa, M, et. al, Biochim. Biophys. Acta, 14:151-160, 1999-   6. Hincke, M. T., et. al, Matrix Biology, 19:443-453, 2000-   7. Deal, C L and Moskowitz, R W, Osteoarthritis, 1999. 25:379-395-   8. Matheson, A J and Perry, C M, Drugs Aging. 2003; 20:1041-1060

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. In particular, it is to beunderstood that the present invention contemplates variations in theproteinaceous material, process parameters, including temperature, time,pH, salt (ash), separation techniques, drying or preparation techniques,and proteins of interest.

1. A composition of solubilized proteins obtained from avian eggshellmembrane without the use of proteolytic enzymes or cross-linking agents,wherein the composition is essentially odor-free, has an ash content ofless than 20% per weight of the composition, and comprises a solubilizedprotein content that is at least 80% per weight of the composition,wherein the solubilized protein is Type I collagen, Type V collagen,Type X collagen, elastin, lysozyme, ovotransferrin, desmosine, orB-N-acetylglucosaminidase or a combination thereof.
 2. The compositionof claim 1 wherein the composition further comprises a polysaccharideselected from the group consisting of hyaluronic acid, chondroitin, andglucosamine.
 3. The composition of claim 1, wherein the composition iswater-soluble and is less than about 3 kDa but greater than 400 Da. 4.The composition of claim 1, wherein the composition comprises at leastone activity selected from the group consisting of (1) antioxidantactivity, (2) anti-inflammatory activity, (3) decreasing reactive oxygenspecies formation from animal cells, (4) maintaining mitochondrialfunction under conditions of ROS, (5) decreasing apoptosis undercondition of oxidative stress, (6) decreasing necrosis under conditionof oxidative stress, (7) maintaining cell viability under condition ofoxidative stress, (8) increasing PMN migration, (9) inhibitslipoxygenase activity (9) decreasing PMNB cell migration towardinflammatory mediator leukotriene B4, (10) anti-wrinkling activity, (11)wound healing, and (12) recovery after trauma.
 5. The composition ofclaim 1 wherein the solubilized protein is substantially pure andundenatured.
 6. The composition of claim 1, wherein the compositioncomprises a physiologically acceptable carrier.
 7. The composition ofclaim 1, wherein the composition is formulated for use in medical,cosmetic, dermatological, nutritional or pharmaceutical applications. 8.A composition of solubilized proteins obtained from avian eggshellmembrane without the use of proteolytic enzymes or cross-linking agents,wherein the composition is essentially odor-free, has an ash content ofless than 10% per weight of the composition, comprises about 6% lysine,and comprises a solubilized protein content that is at least 90% perweight of the composition, wherein the solubilized protein is at leastabout 25% collagen and at least about 20% elastin.
 9. The composition ofclaim 8 wherein the composition further comprises a polysaccharideselected from the group consisting of hyaluronic acid, chondroitin, andglucosamine.
 10. The composition of claim 8, wherein the composition iswater-soluble and is less than about 200 kDa but greater than 3 kDa. 11.The composition of claim 8, wherein the composition comprises at leastone activity selected from the group consisting of (1) antioxidantactivity, (2) anti-inflammatory activity, (3) decreasing reactive oxygenspecies formation from animal cells, (4) maintaining mitochondrialfunction under conditions of ROS, (5) decreasing apoptosis undercondition of oxidative stress, (6) decreasing necrosis under conditionof oxidative stress, (7) maintaining cell viability under condition ofoxidative stress, (8) increasing PMN migration, (9) inhibitslipoxygenase activity (9) decreasing PMNB cell migration towardinflammatory mediator leukotriene B4, (10) anti-wrinkling activity, (11)wound healing, and (12) recovery after trauma.
 12. The composition ofclaim 8 wherein the solubilized protein is substantially pure andundenatured.
 13. The composition of claim 8, wherein the compositioncomprises a physiologically acceptable carrier.
 14. The composition ofclaim 8, wherein the composition is formulated for use in medical,cosmetic, dermatological, nutritional or pharmaceutical applications.15. A method of treating a disease, disorder or condition associatedwith oxidative stress comprising administering to said individual aneffective amount of a composition of solubilized eggshell membrane thatis at least 50% protein.
 16. The method of claim 15 wherein thecomposition comprises the composition of claim
 1. 17. The method ofclaim 15 wherein the composition comprises the composition of claim 8.18. The method of claim 15 further comprising treating a disease,disorder or condition associated with oxidative stress from ROS.
 19. Themethod of claim 15 further comprising treating cosmetic conditions ordermatological disorders in an individual.
 20. The method of claim 15further comprising treating an individual for joint health, wounds, orskin conditions.
 21. The method of claim 20 wherein the method istreating a skin condition, the method further comprising topicallyapplying to the skin of an individual an effective amount of acomposition of claim 1 or claim
 8. 22. The method of claim 21, whereinthe skin being treated has less wrinkles, less blocked pores, unevenpigmentation, skin roughness, acne, and more perfusion orvascularization as compared to skin of an individual not receiving thecomposition.
 23. The method of claim 20 wherein the wound has enhancedhealing as compared to a wound in an individual not receiving thecomposition.
 24. The method of claim 20, further comprising treating ajoint in an individual.
 25. The method of claim 24, wherein the joint inis damaged by a disease or condition comprising inflammation, synovitis,inflammatory arthritis, osteoarthritis, osteochondrosis dessicans (OCD),traumatic injury, fractures, surgical trauma or injury, degenerativejoint disease (DJD), septic arthritis or a combination thereof.
 26. Themethod of claim 25 further comprising treating a joint, the methodfurther comprising delivering an effective amount of the compositionlocally to the joint in need of treatment.
 27. The method of claim 26,wherein the individual with the joint being treated has lessmusculoskeletal pain, less musculoskeletal stiffness, increased motionrange, increased mobility as compared to the musculoskeletal pain,musculoskeletal stiffness, motion range, and mobility in an individualwith an untreated joint in need of treatment.
 28. The method of claim 15further comprising treating a disease, disorder or condition associatedwith a mitochondrial disorder in an individual.
 29. The method of claim15 further comprising treating a disease, disorder or conditionassociated with inflammation in an individual.
 30. The method of claim15 comprising administering the composition intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, or continuous infusion to the individual.
 31. Abeverage, food or nutritional product comprising the composition ofclaim 1 or claim
 8. 32. The product of claim 31, which is intended foranimal or human consumption.
 33. A cosmetic, medical, nutritional,dermatological, or pharmaceutical product comprising the composition ofclaim 1 or claim
 8. 34. The product of claim 33, further comprising acosmetically- or pharmaceutically-acceptable carrier.